Mary S. Matsui

Polyphenolic Antioxidant (-)-Epigallocatechin-3-Gallate from Green Tea Reduces UVB-lnduced Inflammatory Responses and Infiltration of Leukocytes in Human Skin

Abstract— Identification of natural products capable of affording protection against UVB radiation‐induced inflammatory responses and generation of oxidative stress may have important human health implications. The UVB exposure‐induced skin injury and oxidative stress has been associated with a variety of skin disease conditions including photoaging, inflammation and cancer. Tea is a popular beverage consumed worldwide. In several mouse skin models, topical application as well as oral consumption of green tea has been shown to afford protection against chemical and UVB‐induced carcinogenesis and inflammatory responses. In the present study, we investigated in human skin, whether topical application of (‐)epigallocatechin‐3‐gallate (EGCG), the major polyphenolic constituent in green tea, inhibits UVB‐induced infiltration of leukocytes (macrophage/neutrophils), a potential source of generation of reactive oxygen species (ROS), and generation of prostaglandin (PG) metabolites. Human subjects were UVB irradiated on sun‐protected skin to four times their minimal erythema dosage (MED) and skin biopsies or keratomes were obtained either 24 h or 48 h later. We found that topical application of EGCG (3 mg/2.5 cm2) before UVB (4 MED) exposure to human skin significantly blocked UVB‐induced infiltration of leukocytes and reduced myeloperoxidase activity. These infiltrating leukocytes are considered to be the major source of generation of ROS. In the same set of experiments we found that topical application of EGCG before UVB exposure decreased UVB‐induced erythema. In additional experiments, we found that microsomes from EGCG pretreated human skin and exposed to UVB, compared to UVB exposure alone, produced significantly reduced PG metabolites, particularly PGE2. The PG metabolites play a critical role in free radical generation and skin tumor promotion in multistage skin carcinogenesis. Careful microscopic examination of skin sections, stained with hematoxylin and eosin, under higher magnification (x400) also revealed that EGCG pretreated and UVB exposed human skin contained fewer dead cells in the epidermis with comparison to nonpretreated UVB‐exposed skin. Taken together, our data demonstrate that EGCG has the potential to block the UVB‐induced infiltration of leukocytes and the subsequent generation of ROS in human skin. This may explain the possible mechanism involved in anti‐inflammatory effects of green tea. We suggest that EGCG may be useful as a topical agent for protection against UVB‐induced ROS‐associated inflammatory dermatoses, photoaging and photocarcinogenesis. Further studies are warranted in this direction.

Airborne particle exposure and extrinsic skin aging

For decades, extrinsic skin aging has been known to result from chronic exposure to solar radiation and, more recently, to tobacco smoke. In this study, we have assessed the influence of air pollution on skin aging in 400 Caucasian women aged 70-80 years. Skin aging was clinically assessed by means of SCINEXA (score of intrinsic and extrinsic skin aging), a validated skin aging score. Traffic-related exposure at the place of residence was determined by traffic particle emissions and by estimation of soot in fine dust. Exposure to background particle concentration was determined by measurements of ambient particles at fixed monitoring sites. The impact of air pollution on skin aging was analyzed by linear and logistic regression and adjusted for potential confounding variables. Air pollution exposure was significantly correlated to extrinsic skin aging signs, in particular to pigment spots and less pronounced to wrinkles. An increase in soot (per 0.5 × 10(-5) per m) and particles from traffic (per 475  kg per year and square km) was associated with 20% more pigment spots on forehead and cheeks. Background particle pollution, which was measured in low residential areas of the cities without busy traffic and therefore is not directly attributable to traffic but rather to other sources of particles, was also positively correlated to pigment spots on face. These results indicate that particle pollution might influence skin aging as well.

Pomegranate Fruit Extract Modulates UV‐B–mediated Phosphorylation of Mitogen‐activated Protein Kinases and Activation of Nuclear Factor Kappa B in Normal Human Epidermal Keratinocytes¶

Abstract Excessive exposure of solar ultraviolet (UV) radiation, particularly its UV-B component, to humans causes many adverse effects that include erythema, hyperplasia, hyperpigmentation, immunosuppression, photoaging and skin cancer. In recent years, there is increasing use of botanical agents in skin care products. Pomegranate derived from the tree Punica granatum contains anthocyanins (such as delphinidin, cyanidin and pelargonidin) and hydrolyzable tannins (such as punicalin, pedunculagin, punicalagin, gallagic and ellagic acid esters of glucose) and possesses strong antioxidant and anti-inflammatory properties. Recently, we have shown that pomegranate fruit extract (PFE) possesses antitumor promoting effects in a mouse model of chemical carcinogenesis. To begin to establish the effect of PFE for humans in this study, we determined its effect on UV-B–induced adverse effects in normal human epidermal keratinocytes (NHEK). We first assessed the effect of PFE on UV-B–mediated phosphorylation of mitogen-activated protein kinases (MAPK) pathway in NHEK. Immunoblot analysis demonstrated that the treatment of NHEK with PFE (10–40 μg/mL) for 24 h before UV-B (40 mJ/cm2) exposure dose dependently inhibited UV-B–mediated phosphorylation of ERKl/2, JNK1/2 and p38 protein. We also observed that PFE (20 μg/mL) inhibited UV-B–mediated phosphorylation of MAPK in a time-dependent manner. Furthermore, in dose- and time-dependent studies, we evaluated the effect of PFE on UV-B–mediated activation of nuclear factor kappa B (NF-κB) pathway. Using Western blot analysis, we found that PFE treatment of NHEK resulted in a dose- and time-dependent inhibition of UV-B–mediated degradation and phosphorylation of IκBα and activation of IKKα. Using immunoblot analysis, enzyme-linked immunosorbent assay and electrophoretic mobility shift assay, we found that PFE treatment to NHEK resulted in a dose- and time-dependent inhibition of UV-B–mediated nuclear translocation and phosphorylation of NF-κB/p65 at Ser536. Taken together, our data shows that PFE protects against the adverse effects of UV-B radiation by inhibiting UV-B–induced modulations of NF-κB and MAPK pathways and provides a molecular basis for the photochemopreventive effects of PFE.

Sulfated polysaccharides from red microalgae have antiinflammatory properties in vitro and in vivo.

The primary goal of the present research was to determine whether sulfated polysaccharides derived from red microalgae possess anti inflammatory properties when directed against specific parameters of human skin inflammation. These unique biopolymers were studied in both in vitro and in vivo models of skin inflammation. Human subjects were recruited to participatein a study in which the polysaccharide material was applied topically and shown to inhibit cutaneousery thema induced by a known irritant. Leukocyte migration from capillary blood intosites of inflammation is an essential component of the inflammatory process and occurs in a series of steps, two of which are adhesion and chemotaxis. In vitro, the polysaccharide material primarily inhibited the migration of polymorphonuclear leukocytes (PMNs) toward a standard chemoattractant molecule and also partially blocked adhesion of PMNs to endothelial cells. The data obtained strongly suggest that sulfated polysaccharides derived from red microalgae have significant beneficial potential for use in topical products. In addition, the data suggested that the anti inflammatory mechanism for the polysaccharide was, at least in part, due to inhibition of circulating immune cell recruitment toward inflammatory stimuli.

Kinetics of UV light-induced cyclobutane pyrimidine dimers in human skin in vivo: an immunohistochemical analysis of both epidermis and dermis.

Abstract It is well known that UV exposure of human skin induces DNA damage, and the cumulative effect of such repeated damage is an important contributor to the development of skin cancer. Here, we demonstrate UV dose- and time-dependent induction of DNA damage in the form of cyclobutane pyrimidine dimers (CPD) in skin cells following a single exposure of human skin to UV radiation. CPD+ cells were identified by an immunohistochemical technique using monoclonal antibodies to thymine dimers. The percentage of CPD+ cells was UV dose–dependent, even a suberythemal (0.5 minimal erythemal dose [MED]) dose resulted in detectable level of cells that contained pyrimidine dimers. Forty-eight hours after irradiation the percent of total epidermal cells positive for CPD ranged from 19 ± 8, 36 ± 10, 57 ± 12 and 80 ± 10, and total percent dermal cells positive for CPD ranged from 1 ± 1, 7 ± 3, 16 ± 3 and 20 ± 5, respectively, following 0.5, 1.0, 2.0 and 4.0 MED. CPD were also observed in deeper reticular dermis, which suggest the penetrating ability of UV radiation into the skin. The change in CPD+ cells from 0.5 to 240 h post-UV exposure in both epidermal and dermal compartments of the skin was also quantitated. CPD+ cells were observed in skin biopsies at early time points after UV exposure which remained elevated for 48 h, then declined significantly by 3 days post-UV. A close examination of the skin at and after 3 days following UV exposure indicates the significant removal of DNA damaged cells from the epidermis. Ten days after UV exposure the levels of CPD+ cells in both epidermis and dermis were not significantly different from that in unirradiated skin.

Topical application of green and white tea extracts provides protection from solar-simulated ultraviolet light in human skin

Background:  Tea polyphenols have been found to exert beneficial effects on the skin via their antioxidant properties.

Green Tea Phenol Extracts Reduce UVB‐induced DNA Damage in Human Cells via Interleukin‐12†

Green tea chemoprevention has been a focus of recent research, as a polyphenolic fraction from green tea (GTP) has been suggested to prevent UV radiation‐induced skin cancer. Recently, it was demonstrated that GTP reduced the risk for skin cancer in a murine photocarcinogenesis model. This was accompanied by a reduction in UV‐induced DNA damage. These effects appeared to be mediated via interleukin (IL)‐12, which was previously shown to induce DNA repair. Therefore, we studied whether GTP induction of IL‐12 and DNA repair could also be observed in human cells. KB cells and normal human keratinocytes were exposed to GTP 5 h before and after UVB. UVB‐induced apoptosis was reduced in UVB‐exposed cells treated with GTP. GTP induced the secretion of IL‐12 in keratinocytes. The reduction in UV‐induced cell death by GTP was almost completely reversed upon addition of an anti‐IL‐12‐antibody, indicating that the reduction of UV‐induced cell death by GTP is mediated via IL‐12. The ability of IL‐12 to reduce DNA damage and sunburn cells was confirmed in “human living skin equivalent” models. Hence the previously reported UV‐protective effects of GTP appear to be mediated in human cells via IL‐12, most likely through induction of DNA repair.

Melanosomes Are Transferred from Melanocytes to Keratinocytes through the Processes of Packaging, Release, Uptake, and Dispersion

Recent studies have described the role of shedding vesicles as physiological conveyers of intracellular components between neighboring cells. Here we report that melanosomes are one example of shedding vesicle cargo, but are processed by a previously unreported mechanism. Pigment globules were observed to be connected to the filopodia of melanocyte dendrites, which have previously been shown to be conduits for melanosomes. Pigment globules containing multiple melanosomes were released from various areas of the dendrites of normal human melanocytes derived from darkly pigmented skin. The globules were then captured by the microvilli of normal human keratinocytes, also derived from darkly pigmented skin, which incorporated them in a protease-activated receptor-2 (PAR-2)-dependent manner. After the pigment globules were ingested by the keratinocytes, the membrane that surrounded each melanosome cluster was gradually degraded, and the individual melanosomes then spread into the cytosol and were distributed primarily in the perinuclear area of each keratinocyte. These results suggest a melanosome transfer pathway wherein melanosomes are transferred from melanocytes to keratinocytes via the shedding vesicle system. This packaging system generates pigment globules containing multiple melanosomes in a unique manner.

Non-Sunscreen Photoprotection: Antioxidants Add Value to a Sunscreen

The association between ultraviolet radiation (UVR) exposure and both skin cancer and photo-aging is well documented. In addition to the conventional organic-chemical and physical-mineral type sunscreens, other non-sunscreen protective strategies have been developed. These include topically applied botanical extracts and other antioxidants as well as topical DNA repair enzymes. Standard terms of photoprotection such as sun protection factor (SPF) do not accurately reflect the photoprotection benefits of these materials. For example, in spite of minimal SPF, tea extract containing polyphenols such as (-)-epigallocatechin-3-gallate (EGCG) has been shown to protect against UV-induced DNA damage and immune suppression, in part through its ability to reduce oxidative stress and inhibit NF-kB. The addition of botanical antioxidants and vitamins C and E to a broad-spectrum sunscreen may further decrease UV-induced damage compared with sunscreen alone. These agents have been shown to enhance protection against UV-induced epidermal thickening, overexpression of MMP-1and MMP-9, and depletion of CD1a(+) Langerhans cells. Non-sunscreen materials such as botanical extracts, antioxidants, and DNA repair enzymes can contribute value when applied topically to human skin in vivo.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 56-59; doi:10.1038/jidsymp.2009.14.

SIRT1 Promotes Differentiation of Normal Human Keratinocytes

Sir2 regulates lifespan in model organisms, which has stimulated interest in understanding human Sir2 homolog functions. The human Sir2 gene family comprises seven members (SIRT1-SIRT7). SIRT1, the human ortholog of the yeast Sir2 by closest sequence similarity, is a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase with enzymatic properties indistinguishable from the yeast enzyme. We studied the involvement of SIRT1 in normal human keratinocyte physiology by a transcriptional microarray analysis of primary keratinocytes either overexpressing or underexpressing SIRT1. Using a systems biology analytical approach, we predicted that SIRT1 induces keratinocyte differentiation through a pathway integral to or overlapping with that of calcium-induced differentiation. We experimentally assayed this prediction and found that the SIRT1 inhibitor nicotinamide inhibited expression of keratinocyte differentiation markers, whereas a SIRT1 activator, resveratrol, enhanced expression of keratinocyte differentiation markers. Similar results were obtained in keratinocytes manipulated to overexpress or underexpress SIRT1, and modulating SIRT1 significantly affected keratinocyte proliferation rates. We conclude that SIRT1 functions in normal human keratinocytes to inhibit proliferation and to promote differentiation.