Thomas Mammone

Gender-linked differences in human skin

BACKGROUND The physiology of body organs can be affected by gender. Skin and skin appendages are influenced by sex hormones. OBJECTIVE This review work has been undertaken to point out the most conspicuous physiological differences observed between mens and womens skin. METHODS The literature has been searched and relevant results have been gathered. RESULTS Mens and womens skins differ in hormone metabolism, hair growth, sweat rate, sebum production, surface pH, fat accumulation, serum leptins, etc. Examples of differences in the proneness to cutaneous diseases and skin cancer are quoted. CONCLUSION The knowledge of gender-linked cutaneous differences might help in preparing male-specific products for more appropriate dermatological treatments or cosmetic interventions.

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.

Apoptotic cell death increases with senescence in normal human dermal fibroblast cultures

Normal human dermal fibroblasts have a limited life‐span in vitro and stop proliferation after a fixed number of cell divisions. This process by which cells stop proliferation is called senescence. Senescence is also characterized by a decrease in the total cell number. In this study, we characterized an increase in cell death in normal human dermal fibroblasts in vitro as a function of increasing cell passage. With increasing passage, human fibroblasts showed an increase in the number of dead cells and increased DNA fragmentation as determined by flow cytometry. Serial passage of human fibroblasts also resulted in mitochondrial dysfunction, represented by a loss of mitochondrial membrane potential. The apoptotic markers caspase‐3 and cytochrome c were both found to increase in senescent cells. These results suggest the activation of an apoptotic pathway within a population of human fibroblasts as a function of cell passage.

UV Protective Effects of DNA Repair Enzymes and RNA Lotion

Solar UV radiation is known to cause immune suppression, believed to be a critical factor in cutaneous carcinogenesis. Although the mechanism is not entirely understood, DNA damage is clearly involved. Sunscreens function by attenuating the UV radiation that reaches the epidermis. However, once DNA damage ensues, repair mechanisms become essential for prevention of malignant transformation. DNA repair enzymes have shown efficacy in reducing cutaneous neoplasms among xeroderma pigmentosum patients. In vitro studies suggest that RNA fragments increase the resistance of human keratinocytes to UVB damage and enhance DNA repair but in vivo data are lacking. This study aimed to determine the effect of topical formulations containing either DNA repair enzymes (Micrococcus luteus) or RNA fragments (UVC‐irradiated rabbit globin mRNA) on UV‐induced local contact hypersensitivity (CHS) suppression in humans as measured in vivo using the contact allergen dinitrochlorobenzene. Immunohistochemistry was also employed in skin biopsies to evaluate the level of thymine dimers after UV. Eighty volunteers completed the CHS portion. A single 0.75 minimum erythema dose (MED) simulated solar radiation exposure resulted in 64% CHS suppression in unprotected subjects compared with unirradiated sensitized controls. In contrast, UV‐induced CHS suppression was reduced to 19% with DNA repair enzymes, and 7% with RNA fragments. Sun protection factor (SPF) testing revealed an SPF of 1 for both formulations, indicating that the observed immune protection cannot be attributed to sunscreen effects. Biopsies from an additional nine volunteers showed an 18% decrease in thymine dimers by both DNA repair enzymes and RNA fragments, relative to unprotected UV‐irradiated skin. These results suggest that RNA fragments may be useful as a photoprotective agent with in vivo effects comparable to DNA repair enzymes.

Cigarette smoke-induced lipid peroxidation in human skin and its inhibition by topically applied antioxidants.

Cigarette smoke, whether indirect or direct stream, is an environmental pollutant which presents an increasing health problem. In order to determine damage to human skin at the biochemical level, volar forearms were exposed to cigarette smoke for fifteen minutes and then assayed for the presence of stratum corneum lipid peroxides. A time-dependent increase was observed over a 24-hour post-exposure period. At 24 h, the average baseline level of lipid peroxides was 14.9 nmol/unit area of skin as compared to 32.0 nmol/unit area of skin for the smoke-exposed arms. In addition, when topical antioxidants were pre-applied to the skin and then exposed to cigarette smoke, an average decrease of 40.9% in lipid peroxide values was observed. These data demonstrate that peroxidation was induced in human skin by cigarette smoke and subsequently inhibited by the presence of antioxidants.

The Induction of Terminal Differentiation Markers by the cAMP Pathway in Human HaCaT Keratinocytes

The terminal differentiation of human epidermal keratinocytes is a complex morphological and biochemical shift from a mitotically active cell to an inert protein cross-linked envelope. This transition is a clearly predetermined cell death mechanism, but it is unlike many other programmed cell deaths in that it is not apoptotic. To explore and contrast the mechanism by which keratinocytes are committed to differentiation rather than apoptosis, we focused on the cyclic adenosine monophosphate (cAMP) signaling pathway using selective modulators of intracelluar cAMP levels. Markers of differentiation were assayed by Western blotting. Raising intracelluar cAMP levels by treating HaCaT cells with forskolin, a diterpene, or with isobutylmethylxanthine, a phosphodiesterase inhibitor, and isoproterenol, a β-adrenergic receptor agonist that selectively activates adenylate cyclase, increased the levels of the differentiation markers keratin K1 and K10, involucrin and transglutaminase. H89 and KT5720, both inhibitors of cAMP-dependent protein kinase, suppressed the expression of keratins K1 and K10. These observations are in line with the defined role for cAMP in the control of keratinocyte differentiation.

Correlation between in vitro Cyclic Adenosine Monophosphate Phosphodiesterase Inhibition and in vivo Anti-Inflammatory Effect

We tested the hypothesis that topical adenosine monophosphate phosphodiesterase (cAMP PDE) inhibitors are anti-inflammatory. These can be shown by a correlation between PDE inhibitory and anti-inflammatory function of a series of known PDE inhibitors. The effect of various cAMP PDE inhibitors on PDEs isolated from HaCaT cells was first investigated. These compounds were then tested as anti-irritants against topical 8% Balsam of Peru. A direct correlation was observed between the in vitro EC50 values for PDE inhibition and the in vivo anti-inflammatory potential with a correlation coefficient of r = 0.79. These results stress the value of PDE inhibitors as anti-inflammatory agents in topical use, and also demonstrate that the in vitro PDE assay can be used to predict in vivo anti-inflammatory potential.

Modification of skin discoloration by a topical treatment containing an extract of Dianella ensifolia: a potent antioxidant

Skin hyperpigmentation, and the reactions that precipitate it, have been linked to free radicals by the fact that free radical scavengers or antioxidants can slow that hyperpigmentation. We have screened several hundred plant extracts for antioxidants and discovered one that is both a strong antioxidant and can reduce skin hyperpigmentation. Extracts of Dianella ensifolia contain 1‐(2,4‐dihydrophenyl)‐3‐(2,4‐dimethoxy‐3‐methylphenyl) propane (DP), which was found to inhibit the free radical 1‐1‐diphenyl‐2‐picryl‐hydrazyl (DPPH) with an EC50 value of 78 μm. DP was also found to inhibit Ultraviolet (UV)C‐induced lipid oxidation with an EC50 of about 30 μm. We next investigated the effects of this antioxidant on skin hyperpigmentation. The reduction of discoloration by different topical treatments has been assessed in human volunteers using an in vivo assay for the rate of fading of UVB‐induced tan. Two pharmaceutical formulas containing 4% hydroquinone (HQ) were used as positive controls, and we tested the ability of DP, a plant‐derived amphoteric antioxidant, to increase performance of non‐HQ cosmetic formulations. We found that the cosmetic formula containing DP produced an increase in the rate of fading compared to the two pharmaceutical treatments containing HQ.

The Cytoprotective Effects of Exogenous DNA Fragments

Ultraviolet irradiation of normal human keratinocytes induces a cytotoxic effect. The chromophore for this effect is believed to be genomic DNA. However, DNA damage is known to be repaired in UVB irradiated keratinocytes. The trigger for this DNA repair is potentially damaged DNA itself. To test the hypothesis that damaged DNA can induce the host cell’s own DNA repair mechanism, we treated the keratinocytes with the damaged DNA and evaluated its cytoprotective effects. We have observed that fragmented calf thymus DNA irradiated and damaged with a UVC light can induce a protective effect in cultured human keratinocytes. Keratinocytes treated with UVC damaged DNA fragments are less susceptible to UVB irradiation-induced cell death as measured by neutral red uptake. Unirradiated exogenous DNA did not induce this protective effect. Similar protective effects can be seen with irradiated salmon sperm DNA. UVC damaged DNA fragments induced 60% increase in protection in human HaCaT keratinocyte in culture to the cell death induced by UVB. Similar protection was observed with UVC irradiated oligothymidylic acid (dT3-dT5) which increased the survival of human HaCaT keratinocytes after UVB irradiated by 50%. Isolated mononucleotides, irradiated or not, do not increase UVB survivability. Cellular DNA synthesis was greatly inhibited by UVB, becoming undetectable at 40 mJ/cm2. Exogenous treatment with damaged fragments causes immediate and significant inhibition of total cellular DNA synthesis. This inhibition was dose dependent. Cells that undergo damage to their DNA are known to inhibit endogenous DNA synthesis via p53 suppressor gene activation. This is believed to allow them sufficient time to repair the host DNA. The cellular response to exogenous damaged DNA may be a similar mechanism.