Hitoshi Masaki

Role of antioxidants in the skin: Anti-aging effects

Intracellular and extracellular oxidative stress initiated by reactive oxygen species (ROS) advance skin aging, which is characterized by wrinkles and atypical pigmentation. Because UV enhances ROS generation in cells, skin aging is usually discussed in relation to UV exposure. The use of antioxidants is an effective approach to prevent symptoms related to photo-induced aging of the skin. In this review, the mechanisms of ROS generation and ROS elimination in the body are summarized. The effects of ROS generated in the skin and the roles of ROS in altering the skin are also discussed. In addition, the effects of representative antioxidants on the skin are summarized with a focus on skin aging.

UV-induced DNA damage initiates release of MMP-1 in human skin.

Abstract:  Destruction of collagen is a hallmark of photoaging. The major enzyme responsible for collagen 1 digestion, matrix metalloproteinase‐1 (MMP‐1), is induced by exposure to sunlight. To study the molecular trigger for this induction, human skin was ultraviolet‐B (UVB)‐irradiated and treated with liposome‐encapsulated DNA repair enzymes. The photolyase‐mediated DNA repair of epidermal UV damage was associated with a reduction of MMP‐1 mRNA and protein expression in both the epidermal and dermal compartments of the skin. The role of the epidermal cells in MMP‐1 induction in the fibroblasts was examined when human epidermal keratinocytes were irradiated with UVB and their media were transferred to unirradiated human dermal fibroblasts. Transfer of media from irradiated keratinocytes to unirradiated fibroblasts enhanced MMP‐1 mRNA and protein. Thus, UV damage to keratinocytes of the epidermis may participate in the destruction of collagen in the dermis by release of soluble mediators that signal fibroblasts to release MMP‐1. The MMP‐1 induction was reduced when the keratinocytes were treated with DNA repair enzymes T4 endonuclease V or UV endonuclease prior to transfer of the media to fibroblasts. This implies that UVB, which deposits most of its energy on the chromatin of the epidermal keratinocytes and to a lesser extent in the upper dermis, has a significant role in photoaging. DNA damage in the keratinocytes initiates one of the signals for MMP‐1 release, and enhancing DNA repair can reduce MMP‐1 expression in human skin cells and tissue.

Generation of active oxygen species from advanced glycation end-products (AGEs) during ultraviolet light A (UVA) irradiation and a possible mechanism for cell damaging

Advanced glycation end-products (AGEs) have been reported to be accumulated in dermal skin. However, the role of AGEs in the photoaging of human skin remains unknown, and for this reason, we have examined the interaction between AGEs and ultraviolet A light (UVA) from both the chemical and biological aspects. Previously, we reported that exposing human dermal fibroblasts to UVA in the presence of AGEs that were prepared with bovine serum albumin (BSA) decreased the cell viability due to superoxide anion radical s (.O2(-)) and hydroxyl radicals (.OH) generated by AGEs under UVA irradiation, and active oxygen species are detected with ESR spin-trapping. To identify the active oxygen species in detail and to clarify the cell damaging mechanism, we performed several experiments and the following results were obtained. (1) In ESR spin-trapping, by addition of dimethyl sulfoxide and superoxide dismutase, ESR signals due to .O2(-) -derived DMPO-OOH and .OH-derived DMPO-OH adducts, respectively, were detectable. (2) UVA-irradiated AGEs elevated the lipid peroxide levels in both fibroblasts and liposomes. But the peroxidation in liposomes was inhibited by addition of deferoxamine. (3) Survival of fibroblasts exposed to UVA in the presence of AGEs was elevated by addition of deferoxamine. And finally, (4) survival of fibroblasts was found to be regulated by the level of H2O2. On the basis of these results, we propose a possible mechanism in which AGEs under UVA irradiation generate active oxygen species involving .O2(-), H2O2, and .OH, and the .OH species plays a harmful role in promoting cell damage.

Increased generation of hydrogen peroxide possibly from mitochondrial respiratory chain after UVB irradiation of murine fibroblasts

The purpose of this study is to detect the generation of active oxygens in UVB-irradiated murine fibroblasts and to propose new mechanisms. Decreased survival of fibroblasts under UVB irradiation was partially recovered by addition of catalase, DMSO or deferoxamine, suggesting the contribution of several types of active oxygen species. Then we examined the formation of active oxygen species and found that fibroblasts under UVB irradiation generated superoxide anion radicals (.O2-), intracellular H2O2, and hydroxyl radicals as estimated by the ESR-spin trapping method. Addition of thenoyltrifluoroacetone, which is an inhibitor of the mitochondrial respiratory chain, decreased 29% of the intracellular H2O2 levels in UVB-irradiated cells, but allopurinol, which is an inhibitor of xanthine oxidase, had no effect on them. On the basis of these results, we propose a a possible mechanism for damage of murine fibroblasts exposed to UVB in terms of generation of active oxygen species. The mitochondrial respiratory chain reaction stimulated by UVB irradiation enhances the generation of .O2-, which is in turn dismutated to H2O2 and O2 by superoxide dismutase. H2O2 is then converted to hydroxyl radicals, catalyzed by trace elements such as iron, as suggested by Fenton-like reaction. Thus, hydroxyl radicals with higher reaction rate-constants than those of other active oxygen species to biomolecules are indicated to be responsible for the cytotoxicity in cells under UV irradiation.

Reactive Oxygen Species in HaCaT Keratinocytes After UVB Irradiation Are Triggered by Intracellular Ca2+ Levels

It is recognized that reactive oxygen species (ROS) are responsible for skin damage due to UVB-radiation (UVB-R). However, the triggering substance(s) for ROS generation after UVB-R is uncertain with respect to the activation of NADPH oxidase (Nox), xanthine oxidase (XOD), and respiratory chain-chain reactions in mitochondria. As a first step in identifying the trigger(s) for UVB-induced ROS generation, we examined the relationship between Ca(2+) levels and ROS generation in HaCaT keratinocytes. UVB-R exposure of HaCaT keratinocytes resulted in an immediate elevation of ROS that recurred 7 hours later. This was accompanied by immediately elevated intracellular Ca(2+) . A Ca(2+) chelating agent, BAPTA, abolished the elevation of ROS after UVB-R completely. In addition, exogenous H(2)O(2) did not increase intracellular Ca(2+) levels. This suggests that intracellular Ca(2+) is the first trigger for UVB-induced ROS generation.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 50-52; doi:10.1038/jidsymp.2009.12.

α-Tocopherol Increases the Intracellular Glutathione Level in HaCaT Keratinocytes

f -Tocopherol is a lipophilic vitamin that exhibits an antioxidative activity. The purpose of this study was to clarify the roles of f -tocopherol in the regulation of intracellular glutathione (GSH) levels in HaCaT keratinocytes. When HaCaT keratinocytes were cultivated with f -tocopherol for 24 h, the intracellular GSH was increased at every concentration of f -tocopherol tested. Furthermore, the HaCaT keratinocytes cultured with f -tocopherol at 50 w M for 24 h exhibited resistance against H 2 O 2 . However, a short exposure of HaCaT keratinocytes to f -tocopherol for 1 h did not influence either the GSH level or the resistance to H 2 O 2 . These findings suggest that GSH, which is inductively synthesized by f -tocopherol, effectively reduces exogenous oxidative stress. To evaluate the effect of f -tocopherol on the GSH level, BSO, which is a typical inhibitor of n -glutamylcysteine synthetase ( n -GCS), was used. When BSO was added to HaCaT keratinocytes, no action of f -tocopherol on the GSH level was observed. On the other hand, f -tocopherol resulted in the up-regulation of n -GCS-HS (heavy subunit) mRNA. In addition, water soluble f -tocopherol derivatives ( f -tocopherol phosphate and trolox) caused no changes in GSH level. From these results, it was concluded that f -tocopherol increases the intracellular GSH level of HaCaT keratinocytes through the up-regulation of n -GCS-HS mRNA.

Effects of UV irradiation on the sebaceous gland and sebum secretion in hamsters

BACKGROUND Although an understanding of the photobiology of the skin has been extensively advanced recently, the effect of ultraviolet (UV) radiation on sebaceous glands is not well known. OBJECTIVE In this study, we examined the direct effect of UV radiation on cultured sebocytes from hamsters in vitro experimental system. Moreover, we examined whether UV-induced peroxidation of skin surface lipids may affect barrier function of horney layer. METHODS We irradiated cultured sebocytes from hamsters, which have similar biological characteristics to the human sebocytes, with UV radiation. Moreover, transepidermal water loss (TEWL) was examined after topical application of cholesterol or triglyceride (TG) and UV exposures on the back of hamsters. RESULTS The number of sebocytes were increased significantly (120-140%) after 4 days as compared with the non-irradiated controls. Lipid production in sebocytes was also increased on day 7 in an irradiation-dependent manner up to 4.1 times of the pre-irradiated level. When UVB was irradiated to TG- or cholesterol-applied skin at the minimum ear-swelling dose, TEWL increased twice or more as compared with UVB irradiation to unapplied sites. When in vitro-irradiated TG, in vitro-irradiated cholesterol, TG-peroxide (TG-OOH), and cholesterol-peroxide (CHO-OOH) were applied to the skin, TEWL increased significantly. CONCLUSION These results suggest that UVB may directly activate the functions of the sebaceous gland in vivo to produce increased amounts of sebum, which may undergo peroxidation by UV light and damage the barrier functions of the skin.

Hamamelitannin as a new potent active oxygen scavenger

Abstract The active oxygen scavenging activities of hamamelitannin (2′, 5-di- O -galloylhamamelose), which is a major constituent of Hamamelis virginiana , gallic acid, which is thought to be the active moiety of hamamelitannin, and propyl gallate which is a well-known antioxidant, were evaluated. In superoxide anion scavenging, the IC 50 values, which represent the concentration giving 50% inhibition of active oxygens generated, of hamamelitannin (1.31±0.06 μM), gallic acid (1.01±0.03 μM) and propyl gallate (1.41±0.01 μM) were higher than that of ascorbic acid (23.31±2.23 μM). In hydroxyl radical scavenging, hamamelitannin gave the lowest IC 50 value (5.46±0.04 μM) among the compounds tested; the values of gallic acid and propyl gallate were 78.04±11.23 and 86.46±2.31 μM, respectively. In singlet oxygen scavenging, the IC 50 values of hamamelitannin, gallic acid and propyl gallate were 45.51±2.00, 69.81±4.66 and 66.66±2.46 μM, respectively. Hamamelitannin was also found to have antioxidative and scavenging activities against organic radicals like DPPH (1, 1-diphenyl-2-picrylhydrazyl). In a cell culture system, the protective activities of hamamelitannin on cell damage induced by active oxygens, such as superoxide anion radicals, hamamelitannin on cell damage induced by active oxygens, such as superoxide anion radicals, hydroxyl radicals and singlet oxygens, were also evaluated. Against superoxide anion radicals hamamelitannin and propyl gallate protected the cells at over 50 μM, while the protective activity of gallic acid was observed at 500 μM. In contrast, against hydroxyl radicals, hamamelitannin showed protective activity at over 500 μM, while gallic acid and propyl gallate protected at 50 μM. Against singlet oxygens, hamamelitannin indicated survival of 80.6±1.5% at 100 μM, in which the survival of the control was 60.4±6.2%. Gallic acid and propyl gallate showed survivals of 98.6±7.3 and 85.2±4.9% at 500 μM, respectively. DMPO (5,5-dimethyl-1-pyrroline- N -oxide) and PBN (α-phenyl- N-t -butylnitrone) typical ESR spin-trap reagents, were found to protect against cell damage induced by superoxide anion and hydroxyl radicals.

Pentosidine in advanced glycation end-products (AGEs) during UVA irradiation generates active oxygen species and impairs human dermal fibroblasts

Our previous study reported that advanced glycation end-products (AGE)-modified BSA produced active oxygen species, *O2-, H2O2, and *OH under UVA irradiation and enhanced the cytotoxicity of UVA light. We examined whether pentosidine in AGE-modified BSA was involved in one of the mechanisms generating the active oxygen species. In biological investigations, fibroblasts exposed to UVA (20 J/cm2) in the presence of pentosidine-rich compounds (PRCs), which were prepared with L-arginine, L-lysine and glucose, showed a time-dependent leakage of the cytosolic enzyme LDH. In addition, release of LDH was suppressed by addition of DMSO and deferoxamine under UVA irradiation. From these results, it was determined that PRCs exposed to UVA damaged the plasma membrane of human dermal fibroblasts due to the conversion of *OH from H2O2 via a Fenton-like reaction. These features of PRCs exposed to UVA were consistent with those of AGE-modified BSA. In an ESR study, PRCs under UVA irradiation yielded DMPO-OH (DMPO-OH adduct) using DMPO as a spin-trapping reagent. *O2- generation from UVA-irradiated PRCs was also indicated by the combination of NBT reduction and SOD. When PRCs were exposed to UVA light controlled with a long-pass filter, WG-360, it was found that their production of *O2- was prohibited less than 50% in the NBT reduction assay. The *O2- production profile of PRCs depending on the wavelength of UVA light was similar to that of AGE-modified BSA. Furthermore, it was found that the H2O2 level was increased by PRCs exposed to UVA. These results indicated that pentosidine is an important factor of AGE-modified BSA in active oxygen generation under UVA irradiation.

The Mechanism of Melanocytes-Specific Cytotoxicity Induced by Phenol Compounds Having a Prooxidant Effect, relating to the Appearance of Leukoderma

Specific phenol compounds including rhododendrol (RD), a skin-brightening ingredient in cosmetics, are reported to induce leukoderma, inducing a social problem, and the elucidation of mechanism of leukoderma is strongly demanded. This study investigated the relationship among the cytotoxicities of six phenol compounds on B16F10 melanoma cells and HaCaT keratinocytes and generated reactive oxygen species (ROS). As a result, the cytotoxicity of RD on B16F10 cells was higher than that on HaCaT cells, and RD significantly increased intracellular ROS and hydrogen peroxide (H2O2) levels in B16F10 cells. Furthermore, although raspberry ketone (RK), RD derivative, also increased intracellular ROS in B16F10 cells, increase in ROS was suppressed by disodium dihydrogen ethylenediaminetetraacetate dehydrate (EDTA). The amounts of increased ROS with RK in HaCaT cells without melanocyte were further increased by tyrosinase. Therefore, tyrosinase, a metalloprotein having copper, was speculated to be one of causative agents allowing phenol compounds to work as a prooxidant. Hydroxyl radical was generated by adding a mixture of tyrosinase and H2O2 to RD, and the amount of the radical was further increased by UVB, indicating that RD cytotoxicity was caused by intracellularly increased ROS, which possibly related to phenol induced prooxidants.