Changes of superoxide dismutase, catalase and glutathione peroxidase in the corneal epithelium after UVB rays. Histochemical and biochemical study

In this study, the effects of UVA and UVB rays on antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase) were examined in the corneal epithelium. The corneas of albino rabbits were irradiated with a UV lamp generating UVA (365 nm wavelength) or UVB rays (312 nm wavelength), 1 x daily for 5 min, from a distance of 0.03 m, over 4 days (shorter procedure) or 8 days (longer procedure). In contrast to UVA rays, which did not evoke significant disturbances, UVB rays changed the activities of antioxidant enzymes. The longer repeated irradiation with UVB rays was performed, the deeper the observed decrease in antioxidant enzymes. The shorter procedure evoked a more profound decrease of glutathione peroxidase and catalase (the enzymes cleaving hydrogen peroxide) than of superoxide dismutase, an enzyme scavenging superoxide radical and producing hydrogen peroxide during the dismutation reaction of a superoxide free radical. This may contribute to an insufficient hydrogen peroxide cleavage at the corneal surface and danger to the cornea from oxidative damage. After the longer procedure (UVB rays), the activities of all antioxidant enzymes were very low or completely absent. In conclusion, repeated irradiation of the cornea with UVB rays evokes a deficiency in antioxidant enzymes in the corneal epithelium, which very probably contributes to the damage of the cornea (and possibly also deeper parts of the eye) from UVB rays and the reactive oxygen products generated by them.     

Reactive oxygen species (ROS)-generating oxidases in the normal rabbit cornea and their involvement in the corneal damage evoked by UVB rays

The corneas of albino rabbits were irradiated (5 min exposure once a day) with UVB rays (312 nm) for 4 days (shorter procedure) or 8 days (longer procedure). The eyes were examined microbiologically and only the corneas of sterile eyes or eyes with non-pathogenic microbes were employed. Histochemically, the activities of reactive oxygen species (ROS)-generating oxidases (xanthine oxidase, D-amino acid oxidase and alpha-hydroxy acid oxidase) were examined in cryostat sections of the whole corneas. Biochemically, the activity of xanthine oxidoreductase/xanthine oxidase was investigated in the scraped corneal epithelium. UVB rays significantly changed enzyme activities in the corneas. In comparison to the normal cornea, where of ROS-generating oxidases only xanthine oxidase showed significant activity in the corneal epithelium and endothelium, D-amino acid oxidase was very low and alpha-hydroxy acid oxidase could not be detected at all, in the cornea repeatedly irradiated with UVB rays, increased activities of xanthine oxidase and D-amino acid oxidase were observed in all corneal layers. Only after the longer procedure the xanthine oxidase and D-amino acid oxidase activities were decreased in the thinned epithelium in parallel with its morphological disturbances. Further results show that the xanthine oxidase/xanthine oxidoreductase ratio increased in the epithelium together with the repeated irradiation with UVB rays. This might suggest that xanthine dehydrogenase is converted to xanthine oxidase. However, in comparison to the normal corneal epithelium, the total amount of xanthine oxidoredutase was decreased in the irradiated epithelium. It is presumed that xanthine oxidoreductase might be released extracellularly (into tears) or the enzyme molecules were denatured due to UVB rays (particulary after the longer procedure). Comparative histochemical and biochemical findings suggest that reactive oxygen species-generating oxidases (xanthine oxidase, D-amino acid oxidase) contribute to the corneal damage evoked by UVB rays.     

Central corneal thickness considered an index of corneal hydration of the UVB irradiated rabbit cornea as influenced by UVB absorber

UVB radiation from sunlight induces an acute corneal inflammation, photokeratitis, accompanied by changes in corneal hydration. We employed a method of ultrasonic pachymetry for daily examination of central corneal thickness as an index of corneal hydration of the rabbit cornea repeatedly irradiated by UVB radiation (312 nm, daily dose of 0.25 J/cm(2) during three or four days) as influenced by UVB absorber (actinoquinol combined with hyaluronic acid) dropped on the ocular surface during irradiation. One day after the third irradiation procedure the animals were sacrificed and corneas examined immuno-histochemically for peroxynitrite formation, a marker of oxidative damage, the antioxidant aldehyde dehydrogenase 3A1 and endothelial nitric oxide synthase, an enzyme generated nitric oxide. Results show that UV absorber combined with hyaluronic acid protected the cornea against UVB-induced changes in corneal thickness and microscopical disturbances to the cornea (both seen after buffered saline application) until the fourth experimental day. These UVB doses are equivalent to a daily exposure of 2.5 hrs of the human cornea to solar UVB radiation for three consecutive days. It is suggested that actinoquinol/ hyaluronic acid drops might be helpful for the human eye in the defence against photooxidative and other oxidative processes.     

Irradiation of the rabbit cornea with UVB rays stimulates the expression of nitric oxide synthases-generated nitric oxide and the formation of cytotoxic nitrogen-related oxidants

Until now, the role of nitric oxide (NO) in cornea irradiated with UVB rays remains unknown. Therefore, we investigated nitric oxide synthase isomers (NOS), enzymes that generate NO, nitrotyrosine (NT), a cytotoxic byproduct of NO, and malondialdehyde (MDA), a byproduct of lipid peroxidation, in rabbit corneas repeatedly irradiated with UVB rays (312 nm, 1x daily for 6 days, the dose per day 1.01 J/cm2) using immunohistochemical methods. The biochemical measurement of nitrite and nitrate has been used for the indirect investigation of NO concentration in the aqueous humor. Results show that in contrast to normal corneas, where of the NOS isomers only endothelial nitric oxide synthase (NOS3) was expressed in a significant amount (in the epithelium and endothelium), in irradiated corneas all NOS isomers (also brain nitric oxide synthase, NOS1, and inducible nitric oxide synthase, NOS2) as well as an indirect measure of ONOO-formation and MDA were gradually expressed, first in the epithelium, the endothelium and the keratocytes beneath the epithelium and finally in the cells of all corneal layers and the inflammatory cells that invaded the corneal stroma. This was accompanied by an elevated concentration of NO in the aqueous humor. In conclusion, repeated irradiation with UVB rays evoked the stimulation of NO production, peroxynitrite formation (demonstrated by NT residues) and lipid peroxidation (evaluated by MDA staining).     

Trehalose treatment accelerates the healing of UVB-irradiated corneas. Comparative immunohistochemical studies on corneal cryostat sections and corneal impression cytology

The UVB-irradiated cornea is damaged by oxidative stress. Toxic oxygen products induced by UVB radiation in the cornea are insufficiently removed by antioxidants, whose numbers decrease with increasing UVB irradiation. In addition, the UVB-irradiated cornea suffers from hypoxic conditions because damaged corneal cells cannot utilize oxygen normally, although the supply of oxygen to the cornea is unchanged (normal). This contributes to attenuated re-epithelialization, corneal neovascularization and apoptotic cell death. Our previous publications reported that trehalose applied on the corneal surface during irradiation significantly suppressed UVB-induced corneal oxidative damage. The results of this study provide for the first time important evidence that trehalose applied on the surface of corneas for two weeks following repeated UVB irradiation (312 nm, daily dose 0.5 J/cm2) accelerated corneal healing, restored corneal transparency and suppressed corneal neovascularization. Compared to buffered saline treatment, following which caspase-3, nitrotyrosine, malondialdehyde and urokinase-type plasminogen activator were still strongly expressed in the corneal epithelium two weeks after irradiation and corneal neovascularization was evident, apoptotic cell death was already significantly reduced after one week of trehalose application. The expression of other markers of injury returned to normal levels during two weeks of trehalose treatment. In conclusion, our results show that trehalose accelerated healing of the UVB irradiated cornea, very probably via suppression of hypoxia-response injury. In addition, immunohistochemical results on corneal cryostat sections corresponded with those obtained using corneal impression cytologies, thus confirming that corneal impression cytologies are useful for diagnostic purposes.     

Dipeptidyl peptidase IV (DPPIV) activity in the tear fluid as an indicator of the severity of corneal injury: a histochemical and biochemical study

Comparative histochemical and biochemical studies on the catalytically active protease Dipeptidyl peptidase IV (DPPIV), have been performed in the rabbit cornea and the tear fluid using a sensitive fluorogenic substrate, Gly-Pro-7-amino-4-Trifluoromethyl Coumarine (AFC). In both normal and experimentally injured corneas, DPPIV activity was detected histochemically and in the tear fluid biochemically. In contrast to the normal cornea where DPPIV activity was absent and in the tear fluid where it was low, during continuous wearing of contact lenses or repeated irradiation of the cornea with UVB rays, slight DPPIV activity appeared first in the superficial layers of the corneal epithelium, while later increased activity was present in the whole epithelium. This paralleled elevated DPPIV activity in the tear fluid. Moreover, during continuous contact lens wear, the increased DPPIV activity in the tear fluid was, in many cases, coincidental with the presence of capillaries in the limbal part of the corneal stroma. After severe alkali burns when corneal ulcers appeared, collagen fragments were active for DPPIV, which was associated with high DPPIV activity in the tear fluid. In conclusion, Gly-Pro-AFC was found to be useful for comparative histochemical and biochemical studies on DPPIV activity in the experimentally injured rabbit eye. Using the method of the tear film collection by a short touch of substrate punches to the respective site of the cornea or conjunctiva we can show that in experimental injuries (wearing of contact lenses, irradiation of the cornea with UVB rays), the damaged corneal cells were the main source for DPPIV activity in the tear fluid. It is suggested that the activity of DPPIV measured in the tear fluid might serve as an indicator of early corneal disorders, e.g. corneal vascularization related to contact lens wear.     

Inhibition of microRNA-129-5p expression ameliorates ultraviolet ray-induced corneal epithelial cell injury via upregulation of EGFR

Existing evidence has highlighted the effect of ultraviolet light radiation leading to corneal epithelium impairment. During this study, we aim to investigate the effect of microRNA-129-5p (miR-129-5p) on the wound healing process of corneal epithelial cells (CECs) induced by ultraviolet rays in mice by targeting epidermal growth factor receptor (EGFR). First, mouse models of ultraviolet ray-induced CEC injury were established and intrastromally injected with different mimic, inhibitor, and short interfering RNA (siRNA) to detect the effect of miR-129-5p on CEC injury. Subsequently, the corneal tissues were obtained to detect the antioxidant ability and EGFR-positive expression rate. The dual-luciferase reporter gene assay was used to test whether EGFR could directly target miR-129-5p. To further investigate the specific mechanism of miR-129-5p and EGFR in CEC injury, CECs were cultured and transfected with miR-129-5p mimic, miR-129-5p inhibitor, siRNA-EGFR, and miR-129-5p inhibitor + siRNA-EGFR. miR-129-5p has been proven to directly target EGFR. Inhibition of miR-129-5p is able to increase the antioxidant capacity, EGFR-positive rate and the expressions of EGFR, B-cell lymphoma-2, zonula occluden-1, occludin, and keratinocyte growth factor-2, but decrease the expression of vascular endothelial growth factor, BCL2-associated X protein, interleukin (IL)-1β, and IL-4. Inhibition of miR-129-5p arrests cells at the S and G2 phases and decreases apoptosis. Our study provides evidence stating that inhibiting miR-129-5p and upregulating EGFR could aid in the repair of mice CEC injury induced by ultraviolet radiation. Therefore, inhibition of miR-129-5p might provide a basic theory in the repair of CEC injury caused by ultraviolet rays.     

Protection of epidermal cells against UVB injury by the antioxidant selenium-containing single-chain Fv catalytic antibody

The antioxidant effect of selenium-containing single-chain Fv catalytic antibody (Se-scFv2F3), a new mimic of glutathione peroxidase, was confirmed using a model system in which cultured rat skin epidermal cells were injured by ultraviolet B (UVB). The cell damage was characterized in terms of lipid peroxidation of the cells, cell viability, and cell membrane integrity. The injury effects of UVB and protection effects of Se-scFv2F3 on the cells were studied using the model system. UVB can damage the cells severely. Upon precultivation of the cells with 0.4U/ml Se-scFv2F3, however, the damage was significantly reduced as shown by the increase in cell viability, the decrease in the malondialdehyde and hydrogen peroxide levels, and the normalization of lactate dehydrogenase activity. In addition, a novel finding that Se-scFv2F3 can stimulate cultured epidermal cells to proliferate under certain conditions was observed.     

Protection of thymosin beta-4 on corneal endothelial cells from UVB-induced apoptosis

Cornea absorbs most of daily ultraviolet (UV) light. An excess of UV damages results in not only keratopathy and cataract but also maculopathy. It has been reported that thymosin beta-4 (Tbeta4) promotes wound healing, decreases inflammatory response and prevents apoptosis of corneal epithelial cells. However, it is not clear whether Tbeta4 protects UVB-induced corneal injury, particularly in corneal endothelial cells because of its non-proliferation in nature. The purpose of this study is to compare the protective effects of Tbeta4 on bovine corneal endothelial (BCE) cells from low- and high-dose UVB damage. In this study, 1 microg/ml of Tbeta4 was added to BCE cells 2 h before low (12.5 mj/cm2) or high dosage (100 mj/cm2) UVB exposure. Using a fluorogenic substrate cleavage assay, we found that Tbeta4 diminished the reactive oxygen species level in BCE cells elicited by UVB. However, the protection of viability by Tbeta4 could only be detected under low-dose UVB exposure. Moreover, both caspase-9 activity and annexin V/propidium iodine staining demonstrated that Tbeta4 only protected BCE cells from low-dose UVB-induced apoptosis but not high-dose UVB-induced necrosis. Together, Tbeta4 protected corneal endothelial cells from UVB-induced oxidative stress and apoptosis after low-dose UVB exposure. The results support further investigation towards topical use or anterior chamber injection of this small hydrophilic peptide in treating and preventing UVB-induced corneal endothelial damage.     

The role of corneal crystallins in the cellular defense mechanisms against oxidative stress

The refracton hypothesis describes the lens and cornea together as a functional unit that provides the proper ocular transparent and refractive properties for the basis of normal vision. Similarities between the lens and corneal crystallins also suggest that both elements of the refracton may also contribute to the antioxidant defenses of the entire eye. The cornea is the primary physical barrier against environmental assault to the eye and functions as a dominant filter of UV radiation. It is routinely exposed to reactive oxygen species (ROS)-generating UV light and molecular O(2) making it a target vulnerable to UV-induced damage. The cornea is equipped with several defensive mechanisms to counteract the deleterious effects of UV-induced oxidative damage. These comprise both non-enzymatic elements that include proteins and low molecular weight compounds (ferritin, glutathione, NAD(P)H, ascorbate and alpha-tocopherol) as well as various enzymes (catalase, glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione reductase, and superoxide dismutase). Several proteins accumulate in the cornea at unusually high concentrations and have been classified as corneal crystallins based on the analogy of these proteins with the abundant taxon-specific lens crystallins. In addition to performing a structural role related to ocular transparency, corneal crystallins may also contribute to the corneal antioxidant systems through a variety of mechanisms including the direct scavenging of free radicals, the production of NAD(P)H, the metabolism and/or detoxification of toxic compounds (i.e. reactive aldehydes), and the direct absorption of UV radiation. In this review, we extend the discussion of the antioxidant defenses of the cornea to include these highly expressed corneal crystallins and address their specific capacities to minimize oxidative damage.     

The molecular mechanism of protecting cells against oxidative stress by 2-selenium-bridged β-cyclodextrin with glutathione peroxidase activity

Ultraviolet B (UVB) induces apoptosis and lipid peroxidation of NIH3T3 cells by producing reactive oxygen species (ROS). Glutathione peroxidase (GPX) is one of the most important antioxidant enzymes in organism and it can scavenge ROS. 2-selenium-bridged β-cyclodextrin (2-SeCD) is a GPX mimic generated in our lab. Its GPX activity is 7.4 U/μmol, which is 7.5 times as much as that of ebselen. In this paper, we have established a cell damage system using UVB radiation. Using this system, we have determined antioxidant effect of 2-SeCD by comparison of malondialdehyde (MDA) and H₂O₂ contents in NIH3T3 cells before and after UVB radiation. Experimental results indicate that 2-SeCD can inhibit lipid peroxidation and protect the cells from the damage generated by UVB radiation. To evaluate the molecular mechanism of this protection, we determined the effect of 2-SeCD on the expression of p53 and Bcl-2 in NIH3T3 cells. The results showed that 2-SeCD inhibits the increase of p53 expression level and the decrease of expression of Bcl-2 induced by UVB radiation. Thus, we have concluded that protection of NIH3T3 cells against oxidative stress by 2-SeCD was carried out by regulation of the expression of Bcl-2 and p53.     

Protective Effect of Carvacrol on Oxidative Stress and Cellular DNA Damage Induced by UVB Irradiation in Human Peripheral Lymphocytes

Exposure to ultraviolet B (UVB; 280‐320 nm) radiation induces the formation of reactive oxygen species (ROS) in the biological system. In this study, we examined the protective effect of carvacrol on UVB‐induced lipid peroxidation and oxidative DNA damage with reference to alterations in cellular an‐tioxidant status in human lymphocytes. A series of in vitro assays (hydroxyl radical, superoxide, nitric oxide, DPPH (2,2‐Diphenyl‐1‐picryl hydrazyl), and ABTS (2,2‐azino‐bis‐3‐ethylbenzothiazoline‐6‐sulfonic acid) radical scavenging assays) demonstrate antioxidant property of carvacrol in our study. UVB exposure significantly increased thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LHPs), % tail DNA and tail moment; decreased % cell viability and antioxidant status in UVB‐irradiated lymphocytes. Treatment with carvacrol 30 min prior to UVB‐exposure resulted in a significant decline of TBARS, LHP, % tail DNA, and tail moment and increased % cell viability as carvacrol concentration increased. UVB irradiated lymphocytes with carvacrol alone (at 10 μg/mL) gave no significant change in cell viability, TBARS, LHP, % tail DNA, and tail moment when compared with normal lymphocytes. On the basis of our results, we conclude that carvacrol, a dietary antioxidant, mediates its protective effect through modulation of UVB‐induced ROS.     

紫外线对荒漠沙蜥皮肤形态、蜕皮、脂质过氧化和抗氧化酶的影响

为了提高体温,荒漠沙蜥喜好晒太阳的同时增加了紫外线对其皮肤的损伤。本实验研究了不同的紫外线强度(110、300、500、800mJ/cm2)对荒漠沙蜥皮肤形态、蜕皮、脂质过氧化和抗氧化酶的影响。结果显示:皮肤损伤和丙二醛含量的最高峰发生在暴露紫外线300、500、800mJ/cm2后的96、48、24h;SOD活性的最低峰发生在暴露紫外线110、300、500、800mJ/cm2后的24、48、12h;CAT活性在暴露紫外线后立即抑制,然后恢复提高。CAT活性的高低往往伴随皮肤的损伤程度和蜕皮的发生,这表明紫外线对皮肤的损伤与皮肤的脂质过氧化密切相关,CAT是一种主要的抗氧化酶。皮肤的角质层对保护皮肤免受紫外线的损伤也有重要作用。     

Alterations in tear biochemistry associated with postanesthetic chronic dry eye syndrome

Perioperative dry eye syndrome (DES) is a common ocular complication of long-term general anesthesia. Chronic DES can lead to permanent damage to the cornea and disturbance of visual function, up to total loss of vision. Here, a relationship between the duration of general anesthesia and the risk of chronic DES in patients was demonstrated. Using an experimental model of perioperative corneal abrasions in rabbits, it was found that introduction of animals to 3-h general anesthesia resulted in clinically significant chronic damage to the cornea in 50% of cases. The development of the complication was not associated with irreversible or long-term impairment of tear secretion, but it was accompanied by a decrease in tear film stability and growth of the total protein content as well as decrease in total antioxidant activity of the tear induced by low molecular weight antioxidants. In addition, anesthesia-induced changes in activity of tear antioxidant enzymes including superoxide dismutase and enzymes providing homeostasis of reduced glutathione (glutathione peroxidase, glutathione-S-transferase, glutathione reductase) were observed. All these alterations were protracted (up to 1-2 weeks) and therefore might account for transition of the perioperative DES into the chronic form. These findings can be useful in the development of novel approaches for the prevention and treatment of chronic forms of DES in the postanesthetic period.     

Photo-protective properties of Lomentaria hakodatensis yendo against ultraviolet B radiation-induced keratinocyte damage

This study aims to investigate the photoprotective properties of a Lomentaria hakodatensis ethanol extract (LHE) against ultraviolet B (UVB) radiation-induced cellular damage in human HaCaT keratinocytes. LHE exhibited scavenging activity against intracellular reactive oxygen species (ROS), which were generated by either hydrogen peroxide (H₂O₂) or UVB radiation. Moreover, LHE scavenged superoxide anion generated by the xanthine/xanthine oxidase system and hydroxyl radical generated by the Fenton reaction (FeSO₄ + H₂O₂). Furthermore, LHE exhibited UVB absorptive properties and attenuated injury to cellular components (e.g., lipids, proteins and DNA), resulting from UVB-induced oxidative stress. In addition, LHE reduced apoptosis in response to UVB, as shown by decreased DNA fragmentation and the formation of apoptotic bodies. These results suggest that LHE protects human keratinocytes against UVB-induced oxidative stress by scavenging ROS and absorbing UVB rays; thereby reducing damage to biological components.     

Differences in activities of antioxidant superoxide dismutase, glutathione peroxidase and prooxidant xanthine oxidoreductase/xanthine oxidase in the normal corneal epithelium of various mammals

Under normal conditions, antioxidants at the corneal surface are balanced with the production of reactive oxygen species without any toxic effects. Danger from oxidative stress appears when natural antioxidants are overwhelmed leading to antioxidant/prooxidant imbalance. The aim of the present study was to examine the activities of enzymes contributing to the antioxidant/prooxidant balance in normal corneal epithelium of various mammals. The enzyme activities of antioxidant superoxide dismutase and glutathione peroxidase, as well as prooxidant xanthine oxidoreductase/xanthine oxidase were examined using biochemical methods. Results show that superoxide dismutase activity is high in rabbits and guinea pigs, whereas in pigs the activity is low and in cows it is nearly absent. In contrast, glutathione peroxidase activity is high in cows, pigs and rabbits, whereas in guinea pigs the activity is low. As far as prooxidant enzymes are concerned, elevated xanthine oxidoreductase/xanthine oxidase activities were found in rabbits, lower activities in guinea pigs, very low activity in cows and no activity in pigs. In conclusion, the above results demonstrate inter-species variations in activities of enzymes participating in antioxidant/prooxidant balance in the corneal epithelium. It is suggested that the levels of antioxidant and prooxidant enzymes studied in the corneal epithelium might be associated with the diurnal or nocturnal activity of animals. UV rays decompose hydrogen peroxide to damaging hydroxyl radicals and perhaps for this reason large animals with diurnal activity (cow, pig) require more effective peroxide removal (high glutathione peroxidase activity) together with the suppression of peroxide production (low superoxide dismutase activity, low xanthine oxidoreductase activity).     

Repeated exposures of human skin equivalent to low doses of ultraviolet-B radiation lead to changes in cellular functions and accumulation of cyclobutane pyrimidine dimers.

Chronic exposure to sunlight may induce skin damage such as photoaging and photocarcinogenesis. These harmful effects are mostly caused by ultraviolet-B (UVB) rays. Yet, less is known about the contribution of low UVB doses to skin damage. The aim of this study was to determine the tissue changes induced by repeated exposure to a suberythemal dose of UVB radiation. Human keratinocytes in monolayer cultures and in skin equivalent were irradiated daily with 8 mJ/cm2 of UVB. Then structural, ultrastructural, and biochemical alterations were evaluated. The results show that exposure to UVB led to a generalized destabilization of the epidermis structure. In irradiated skin equivalents, keratinocytes displayed differentiated morphology and a reduced capacity to proliferate. Ultrastructural analysis revealed, not only unusual aggregation of intermediate filaments, but also disorganized desmosomes and larger mitochondria in basal cells. UVB irradiation also induced the secretion of metalloproteinase-9, which may be responsible for degradation of type IV collagen at the basement membrane. DNA damage analysis showed that both single and repeated exposure to UVB led to formation of (6-4) photoproducts and cyclobutane pyrimidine dimers. Although the (6-4) photoproducts were repaired within 24 h after irradiation, cyclobutane pyrimidine dimers accumulated over the course of the experiment. These studies demonstrate that, even at a suberythemal dose, repeated exposure to UVB causes significant functional and molecular damage to keratinocytes, which might eventually predispose to skin cancer.     

Protective effect of inositol hexaphosphate against UVB damage in HaCaT cells and skin carcinogenesis in SKH1 hairless mice

UVB radiation damages keratinocytes, potentially inducing chronic skin damage, cutaneous malignancy, and suppression of the immune system. Naturally occurring agents have been considered for prevention and treatment of various kinds of cancer, including skin cancer. Inositol hexaphosphate (IP6), an antioxidant, is a naturally occurring polyphosphorylated carbohydrate that has shown a strong anticancer activity in several experimental models. We assessed the protective effects of IP6 against UVB irradiationinduced injury and photocarcinogenesis by using HaCaT cells (human immortalized keratinocytes) and SKH1 hairless mice. We found that IP6 counteracts the harmful effects of UVB irradiation and increases the viability and survival of UVB-exposed cells. Treatment with IP6 after UVB irradiation (30 mJ/cm(2)) arrested cells in the G(1) and G(2) M phases while decreasing the S phase of the cell cycle. Treatment with IP6 also decreased UVB-induced apoptosis and caspase 3 activation. Topical application of IP6 followed by exposure to UVB irradiation in SKH1 hairless mice decreased tumor incidence and multiplicity as compared with control mice. Our results suggest that IP6 protects HaCaT cells from UVB-induced apoptosis and mice from UVB-induced tumors.     

Extracellular superoxide dismutase attenuates release of pulmonary hyaluronan from the extracellular matrix following bleomycin exposure

The major pulmonary antioxidant enzyme involved in the protection of the lung interstitium from oxidative stress is extracellular superoxide dismutase (EC-SOD). It has been previously shown that EC-SOD knock-out mice are more susceptible to bleomycin-induced lung injury, however, the molecular mechanism(s) remains unclear. We report here that bleomycin-induced lung damage, in EC-SOD KO mice, is associated with increased hyaluronan release into alveolar fluid. Analysis of hyaluronan synthase gene expression and hyaluronan molecular weight distribution suggested that elevated levels of hyaluronan in the alveolar fluid are mostly due to its release from the interstitium. Our results indicate that EC-SOD attenuates bleomycin-induced pulmonary injury, at least in part, by preventing superoxide-mediated release of hyaluronan into alveolar space.     

The effects of UV radiation on the visual system of the crab Neohelice granulata: a protective role of melatonin

The first and main target-structure of ultraviolet (UV) radiation in animals is the body surface, including the skin and eyes. Here, we investigated cell damage in the visual system of the crab Neohelice granulata acclimated to constant light and exposed to UVA or UVB at 12:00 h for 30 min. The reactive oxygen species (ROS) production, antioxidant capacity against peroxyl radicals (ACAP), lipid peroxidation (LPO) damage, catalase (CAT) activity, and the melatonin immunohistochemical reactivity in the eyestalks were evaluated. The animals that received melatonin and were exposed to UVA and UVB radiation showed a decreased ROS concentration (p < 0.05).The ACAP test showed a decrease (p < 0.05) in their values when the animals received 2 pmol/crab of melatonin (physiological dose) before the exposure to UVA radiation. The animals exposed to UVB radiation after receiving the same dose of melatonin showed an increase (p < 0.05) in the ACAP test compared with the animals exposed to UVB radiation after receiving only crab physiological saline. The CAT activity increased (p < 0.05) in the animals that received melatonin and were exposed to UVA and UVB radiation. Animals exposed to UVA and UVB displayed an increase (p < 0.05) in the LPO levels, whereas animals treated with melatonin showed lower (p < 0.05) LPO levels when irradiated. The results indicate that the specific oxidative parameters altered by UV radiation can be modulated by a physiological dose of melatonin. Moreover, the melatonin regularly produced by virtually all eyestalk cells suggests that it may function to modulate the noxious effects of radiation, at least in the crab N. granulata.