The effect of N-acetyl-glucosamine on stratum corneum desquamation and water content in human skin.

Alpha-hydroxy acids have been used topically to treat skin for both dermatological and cosmetic problems for many years. Though there are many known benefits of the use of alpha-hydroxy acids on skin, there have been recent reports that topical treatments with alpha-hydroxy acids increase skin damage resulting from UVB. Additionally, high concentrations of alpha-hydroxy acids by themselves have also been found to cause skin irritation. In order to find alternatives to alpha-hydroxy acids, we investigated a variety of amino sugar compounds that were previously reported to inhibit the reaggregation of dissociated corneocytes by modulating cellular adhesion. In vivo, we observed that topical treatments with a formulation containing N-acetyl-glucosamine (NAG) led to an increase in skin moisturization, a decrease in skin flakiness, and the normalization of stratum corneum exfoliation. In vitro, we observed an upregulation of differentiation markers, keratin 10 and involucrin, in keratinocytes treated with NAG. CD44 is a lectin cell adhesion molecule that is also expressed in keratinocytes. Amino sugars such as NAG may competitively bind to CD44, modulating keratinocyte cellular adhesion. We hypothesize that these amino sugars modulate keratinocyte cellular adhesion and differentiation, leading to the normalization of stratum corneum exfoliation. We propose the use of amino sugars such as NAG as alternative compounds to replace the use of alpha-hydroxy acids in skin care.

Psychological stress perturbs epidermal permeability barrier homeostasis: implications for the pathogenesis of stress-associated skin disorders.

BACKGROUND: A large number of skin diseases, including atopic dermatitis and psoriasis, appear to be precipitated or exacerbated by psychological stress. Nevertheless, the specific pathogenic role of psychological stress remains unknown. In 3 different murine models of psychological stress, it was recently shown that psychological stress negatively impacts cutaneous permeability barrier function and that coadministration of tranquilizers blocks this stress-induced deterioration in barrier function. OBJECTIVES AND METHODS: The relationship between psychological stress and epidermal permeability barrier function was investigated in 27 medical, dental, and pharmacy students without coexistent skin disease. Their psychological state was assessed with 2 well-validated measures: the Perceived Stress Scale and the Profile of Mood States. Barrier function was assessed simultaneously with the stress measures at periods of presumed higher stress (during final examinations) and at 2 assumed, lower stress occasions (after return from winter vacation [approximately 4 weeks before final examinations] and during spring vacation [approximately 4 weeks after final examinations]). RESULTS: The subjects as a group demonstrated a decline in permeability barrier recovery kinetics after barrier disruption by cellophane tape stripping, in parallel with an increase in perceived psychological stress during the higher vs the initial lower stress occasions. During the follow-up, presumed lower stress period, the subjects again displayed lower perceived psychological stress scores and improved permeability barrier recovery kinetics, comparable to those during the initial lower stress period. Moreover, the greatest deterioration in barrier function occurred in those subjects who demonstrated the largest increases in perceived psychological stress. CONCLUSION: These studies provide the first link between psychological status and cutaneous function in humans and suggest a new pathophysiological paradigm, ie, stress-induced derangements in epidermal function as precipitators of inflammatory dermatoses.

Johns Hopkins University, Baltimore, Maryland.

The STEM can be used in one of three modes: 1) to image individual atoms; 2) to measure mass or molecular weight; 3) to collect electron energy loss spectra or x-ray fluorescence data. Heavy atom imaging is used to identify chemical groups in a molecule or macromolecules in an assembly. Specific labels have been developed for bases in nucleic acids. These permit localization of bound proteins on single strand nucleic acids. Pt(gly-L-met)Cl is a specific label for methionine residues of proteins as shown with the SLS aggregate of collagen. Lysine can be labeled as well if first methyl (methyl-thio-acetimidate) is coupled. This labeling procedure permits the localization of individual histones within a nucleosome. Mass determination can be used to answer crucial questions about biological assemblies. This is demonstrated by examples from muscle structure.

Performance of the Neutral Red uptake assay in the COLIPA international validation study on alternatives to the rabbit eye irritation test.

The neutral red uptake (NRU) assay was included as part of the COLIPA international validation trial of in vitro alternatives to the Draize eye irritation test. In a blind trial, 55 substances were tested at four laboratories. Following testing, a prediction of the in vivo Draize modified maximum average score (MMAS) for each substance was made by each laboratory using a prediction model relating mean NR(50) value (concentration causing 50% reduction in NRU from that of untreated control cells) to MMAS. Following statistical analysis of the results and breaking of the code, assessment of the results and further analysis was carried out by the participating laboratories. This paper presents the conclusions with regard to the NRU assay. The initial trial analysis indicated that the interlaboratory reproducibility of results of the NRU assay was good. However, there was a poor correlation between observed and predicted MMAS (using the proposed prediction model) when all the test substances were analysed together (r=0.246). Data analysis of subsets of substances indicated that the best predictions were for pure surfactants only (r=0.843) although this data did not fit within the limits of the prediction model. The NRU assay therefore appears to have limited use as a complete Draize replacement. A further examination of the COLIPA trial data may identify combinations of assays which may be more useful than the individual assays which, like NRU, have been shown to be poor predictors of eye irritation.

A summary report of the COLIPA international validation study on alternatives to the draize rabbit eye irritation test.

The principal goal of this study was to determine whether the results from a set of selected currently available alternative methods as used by cosmetics companies are valid for predicting the eye irritation potential of cosmetics formulations and ingredients and, as a consequence, could be valid replacements for the Draize eye irritation test. For the first time in a validation study, prediction models (PMs) that convert the in vitro data from an assay to a prediction of eye irritation were developed for each alternative method before the study began. The PM is an unequivocal description of the relationship between the in vitro and the in vivo data and allows an objective assessment of the reliability and relevance of the alternative methods. In this study, 10 alternative methods were evaluated using 55 test substances selected as representative of substances commonly used in the cosmetics industry (23 ingredients and 32 formulations). Twenty of the single ingredients were common to the European Commission/British Home Office (EC/HO) eye irritation validation study (Balls et al., 1995b). The test substances were coded and supplied to the participating laboratories. The results were collected centrally and analysed independently, using statistical methods that had been agreed before the testing phase began. Each alternative method was then evaluated for reliability and relevance in assessing eye irritation potential. Using the criteria of both reliability and relevance as defined in the study, the preliminary results indicate that none of the alternative methods evaluated could be confirmed as a valid replacement for the Draize eye irritation test across the full irritation scale. However, three alternative methods-the fluorescein leakage test, the red blood cell assay (classification model) and the tissue equivalent assay-each satisfied one criterion of reliability or relevance. Further investigation of the decoded data from this study to explore more fully the relationship between the in vitro data and the in vivo data is recommended. Such a review may allow the development of new prediction models to be tested in a subsequent validation study.

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant-based formulations.

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.

A test for antioxidant activity in cosmetic formulations.

The aim of this study was to develop a technique to assay for the activity of antioxidants in a finished cosmetic product. This was accomplished by adapting the Randox Assay for Total Antioxidant Status kit so that diluted samples could be evaluated by kinetic as well as end-point determinations. Using this technique, we found that a finished product had an IC(50) of 0.07 gm of product and a relative antioxidant activity concentration of 52.7 nmoles/mg.

Normal human keratinocytes pretreated with damaged mRNA are resistant to UVB damage and exhibit increased DNA repair.

Treatment of normal human keratinocytes with UVC-irradiated rabbit globin mRNA 24 h before and after UVB exposure increased the survival of the human keratinocytes. We also observed that UVC-damaged mRNA reduced the formation of sunburn cells in skin models. We next tested the effects of UVC-damaged mRNA on cellular repair of DNA. DNA repair was evaluated using 2 assay methods. The first method used a damaged plasmid that is transfected back into the cell where it is repaired by the host cell repair mechanism. In these experiments, we observed that externally added UVC-damaged rabbit globin mRNA enhanced the repair of a plasmid transfected into the host keratinocyte cells. The second method used to determine the effects on DNA repair was direct immunostaining for thymidine-thymidine dimers (TT dimers) in histological sections of the skin models. Skin models were irradiated with UVB and then fixed immediately or after 24 h and stained for TT dimers. UVB irradiation immediately caused an increase in the number of stained keratinocytes in the skin. The number of stained cells decreased in skin fixed 24 h after UVB. This is due to repair of the TT dimers and their removal. Sections of skin models pretreated with UV-damaged mRNA exhibit greater removal of these TT dimers after 24 h. The above evidence suggests that damaged mRNA can trigger a host cell DNA repair pathway.

Antikeratin antibody staining on ultrathin sections of epidermal cells prepared by low-denaturation embedding.

Direct immunological identification of cellular components has not been possible in tissues prepared for electron microscopy by conventional methods. This may be attributed, in part, to the relatively harsh reagents employed. Using an approach to preparation of biological specimens for transmission electron microscopy that aims for minimal perturbation of native protein conformation, we have obtained specimens that may be stained with antibodies. Recent investigations using these methods have revealed new information regarding the organization of epidermal cells.

Cellulite. Etiology and purported treatment.

Much research must be undertaken before any of the treatments discussed can be validated as clinically effective. At present, it can be safely stated that there is no topical medication or manipulative process to which advanced cellulite visibly responds in a treatment period of less than 2 months.

Discrimination of collagen types by methionine-specific stain.

Chloroglycyl-L-methioninatoplatinum (II) (Pt-GLM) specifically labels methionine residues in collagen. Rat Type I and calf Type III collagen segment long spacing aggregates show differences in Pt-GLM bands, in agreement with known methionine positions. Reconstituted fibers formed from the same two collagens again demonstrate some differences in band pattern, which can be understood in terms of the amino acid sequences and the known packing of molecules in the fiber. Native collagen fibers have been extracted from rat and chick tendon (predominantly Type I collagen). These have been stained with Pt-GLM and studied in the Johns Hopkins STEM and a CTEM. Density profiles along the collagen fibers have been obtained, and those for successive 670-A repeats averaged, by computer programs. The position of peaks in the average density profiles for these various fibers show very good correlation with the known locations of Met residues. The band patterns and density profiles for native and reconstituted Type I fibers are very similar and differ from those of reconstituted Type III fibers.

Factors defining sensitive skin and its treatment.

BACKGROUND: Users of cosmetics and skin care products often report adverse reactions ranging from itching and dryness to intense inflammatory responses such as erythema or wheal and rash. Self-assessment is not always an accurate parameter for categorizing skin as sensitive or nonsensitive, although it can be valuable. For this reason, it is important to define sensitive skin by more objective factors. OBJECTIVE: Studies were undertaken to determine if objective biophysical measurements could detect differences in barrier function between those individuals who identified themselves as having sensitive skin and those self-identified as having normal skin. In addition, the effects of treatment on barrier functions of individuals with sensitive skin were determined. METHODS: Three main factors that contribute to cutaneous reactivities were observed for the estimation of skin sensitivity: barrier functions, reactivity to irritants, and neuronal responses manifested as sensory reactions. Barrier functions of the skin was tested by gentle removal of the stratum corneum with simple cellophane tape stripping followed by measurement of transepidermal water loss (TEWL) as a marker of barrier loss. The onset and intensity of skin reaction against an irritant, balsam of Peru, was tested on the same individuals to observe the reactivity of their skin. Using the lactic acid sting test, additional information regarding skin sensitivities was obtained. RESULTS: Sensitive skin individuals exhibiting easy barrier damage possess delicate skin that is also highly reactive to irritants. When these individuals used a regimen of products that contained minimal preservatives and no surfactants for 8 weeks, the skin barrier and reactivity changed such that it was similar to nonsensitive skin. CONCLUSIONS: Skin sensitivity is observed because of a combination of factors, including a disrupted barrier and a tendency to hyperreact to topical agents. Treatment with special topical skin care formulations can reduce overall skin sensitivity.

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 intracellular 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 beta-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.

Effect of topical application of antioxidants and free radical scavengers on protection of hairless mouse skin exposed to chronic doses of ultraviolet B.

BACKGROUND/AIMS: Within the past three decades, there has emerged a greater awareness of the molecular effects of solar rays especially ultraviolet radiation (UV-R), to the extent that the harmful effects of solar radiation are recognized not only by molecular biologists and physicians, but also by the general public (1). Various sunscreen molecules that effectively block the UVB component of the sun are available; however, a large part of Western populations elicits adverse reactions against chemical sunscreens (2). This study was designed to observe the protective effect of antioxidants against the damaging effects of chronic UVB exposure of skin in an attempt to introduce antioxidants and free radical scavengers as topical sun protective agents. METHODS: Jackson hairless mice were exposed to suberythemal doses of UVB, three times a week, and topically treated with a cream containing the anti-oxidants vitamin E, butylated hydroxy-toluene, nordihydroguaradinic acid and vitamin C. RESULTS: Treatment with vehicle alone along with UVB exposure resulted in an increase in epidermal thickness showing a 38%, 77% and 112% increase after 4 weeks, 8 weeks and 12 weeks, respectively. Chronic UVB exposed skin treated with the material containing free radical scavengers and antioxidants mix (AO mix) exhibited 39%, 73% and 124% thicker epidermis than the un-treated control after, respectively, 4 weeks, 8 weeks and 12 weeks of treatment. The vehicle did not appear to protect skin against UV irradiation, since there appeared to be more (16%) sunburn cells in vehicle treated skin than the untreated, UV exposed skin after 4 weeks of treatment. After 8 weeks and 12 weeks, there were 33% and 36% less sunburn cells in the vehicle treated skin than the untreated, UV exposed skin. The antioxidant mix was significantly effective (P=<0.001) in protecting against UVB irradiation, having 63%, 71 % and 79% fewer sunburn cells than the untreated, UV exposed skin after 4 weeks, 8 weeks and 12 weeks of treatment, respectively. CONCLUSION: Data from these studies suggest that low level chronic exposures to UV can lead to alteration of the skin, like epidermal thickening and appearance of sunburn cells. The data also indicates that a mix of common antioxidants and free radical scavengers are photoprotective against chronic skin damage in the hairless mouse skin model.

Successful separation of apoptosis and necrosis pathways in HaCaT keratinocyte cells induced by UVB irradiation.

UVB irradiation can induce apoptotic, necrotic, and differentiation pathways in normal human keratinocytes. The present study was undertaken to determine at what dose of UVB each of these pathways is induced and whether these pathways are distinct or overlapping. We have observed that UVB induces fragmentation of DNA in human HaCaT keratinocytes, in a bimodal manner. Low doses of UVB, 5-20 mJ/cm2, increase the levels of apoptosis as shown by increased levels of fragmented DNA, Fas, PARP, and FasL protein, and the number of apoptotic cells as assessed by FACS analysis. At higher doses of UVB (20 and 30 mJ/cm2) the number of apoptotic cells becomes reduced, as does the amount of Fas, PARP, and FasL protein. At these higher doses, cell viability is decreased as measured by DNA synthesis (BrdU labeling) neutral red uptake, which represents an increasing necrotic phenotype. Expression of markers of keratinocyte differentiation, involucrin, keratin K1, and keratin K10, are also observed to decrease with increasing UVB dose. These changes are accompanied by a further increase in DNA fragmentation. We conclude that low doses of UVB (5-20 mJ/cm2) induced an apoptotic pathway, whereas increasing doses (greater than 20 mJ/cm2) of UVB produce a direct necrotic effect and inhibit terminal 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 EC(50) 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.

Protection against endogenous and UVB-induced oxidative damage in stratum corneum lipids by an antioxidant-containing cosmetic formulation.

A 16-week human clinical study was carried out to determine the ability of antioxidants in a cosmetic vehicle to inhibit the induction of lipid peroxidation in stratum corneum lipids. The study consisted of a twice daily application of material for 12 weeks followed by a 4-week regression phase. Stratum corneum lipids were collected and then exposed to 500 mJ/cm2 of ultraviolet B (UVB) radiation in order to avoid excessive erythemal damage to the subjects. Lipid peroxides were assayed by a methylene blue derivative assay and expressed per unit area of skin. During the treatment period, decreases in the level of lipid peroxides were observed on the sites treated with the compositions containing antioxidants, as compared to the untreated sites, and expressed as percent differences. Decreases were observed in endogenous as well as UV-induced lipid peroxides followed by a return to baseline levels. These results demonstrate that antioxidants in a topical cosmetic formulation were effective in protecting human stratum corneum lipids against endogenous oxidation or if challenged by 500 mJ/cm2 UVB.

Protection against cigarette smoke-induced damage to intact transformed rabbit corneal cells by N-acetyl-L-cysteine.

In order to assess cigarette smoke-induced oxidative damage to intact cells, an assay was developed to measure cell detachment and protection. Due to the complex nature of cigarette smoke, which contains molecules that can interfere with conventional spectrophotometric and fluorometric biochemical assays, transformed rabbit corneal cells were radiolabeled with tritiated thymidine and then subjected to direct stream smoke. As a result, cell damage in response to the smoke from only two cigarettes could be measured in a time-dependent manner. When cells were prelabeled with N-acetyl-L-cysteine (NAC), a substrate for glutathione synthesis, a significant reduction in damage was measured. Additionally, when buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, was incubated with cells, a reduction in the effectiveness of NAC was observed, although NAC still retained some activity. Furthermore, vitamin E conferred no protection to cells in this system nor was NAC active in a separate assay that appears to favor peroxyl radical generation. From these results we conclude that cigarette smoke damage can easily be determined at the cellular level with this technique and that NAC acted to prevent this damage in two ways: first, as glutathione precursor and, secondly, as an antioxidant capable of scavenging non-peroxyl radicals.

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.