An analytical technique for measuring protein carbonyl in the stratum corneum using surface plasmon resonance

Protein carbonyl groups in the stratum corneum may be used as a biomarker for skin photo-stress. To evaluate the relationship between the protein carbonyl to total protein (TP) ratio (carbonylation ratio) and skin photo-stress, the authors established a methodology by which protein carbonyl can be easily and highly sensitively analysed using an optical technique based on surface plasmon resonance (SPR). To collect the stratum corneum, tape stripping was employed. Firstly, the protein carbonyl was reacted with 2,4-dinitrobenzenesulfonic acid dihydrate, and the quantity of dinitrophenylated (DNP)-protein carbonyl was determined using an anti-dinitrophenyl (anti-DNP) antibody. The mass of DNP-protein carbonyl was measured using SPR. A truncated sampling-reporting cycle of <5 min allowed speedy reporting of DNP-protein carbonyl levels. A significant difference was observed in the protein carbonyl/TP ratio (carbonylation ratio) between a sun-protected area (mid-ventral arm) and a sun-exposed area (upper cheek, P < 0.05). Additionally, the carbonylation ratio of the sun-exposed area showed a higher value than that of the sun-protected area. It was suggested that the carbonylation ratio might be a useful index of skin photo-stress.     

Abstracts: Natural moisturizing factors in the stratum corneum I. Effects of lipid extraction and soaking

pp. 13–22 Natural moisturizing factor (NMF) is essential for appropriate stratum corneum hydration, barrier homeostasis, desquamation, and plasticity. It is formed from filaggrin proteolysis to small, hygroscopic molecules including amino acids. We hypothesized that common lipid extraction and soaking in water would alter the level of NMF in the upper stratum corneum (SC) and its biophysical properties. A novel method of measuring and quantifying the amino acid components of NMF is presented. Adhesive tapes were used to collect samples of the SC and were extracted with 6 mM perchloric acid for analysis by reverse‐phase high‐performance liquid chromatography (HPLC).The HPLC results were standardized to the amount of protein removed by the tapes. An increase in NMF was found with increased SC depth. Also, the combination of extraction and soaking was found to increase NMF loss relative to control or to extraction or soaking alone. Our results indicate that common skin care practices significantly influence the water binding materials in the upper SC. The findings have implications for the evaluation and formulation of skin care products.     

Change in optical properties of stratum corneum induced by protein carbonylation in vitro

The skin is the frontier against the external environment and continuously exposed to the environmental oxidative stress such as ultraviolet (UV) irradiation. Protein carbonyls are the major oxidative products of protein and may be introduced by reaction with aldehydes derived from lipid peroxide. Acrolein is one of the most reactive aldehydes generated during degradation of lipid peroxides and protein-acrolein adducts have been found in the oxidatively damaged lesion including UV-damaged skin. Recent studies revealed that protein carbonyls are also detected in thin outermost layer of the skin, the stratum corneum (SC). However, the effect of protein carbonylation on the function of SC was still unclear. In this study, we treated the SC sheets of reconstructed human epidermis and porcine epidermis with acrolein in the experimental conditions to explore the influence of protein carbonylation on the SC. Human and porcine SC sheets treated with acrolein showed less transmission at visible light than untreated SC sheets. Attenuated total reflection-infrared spectroscopy with curve fitting analysis of amide I region showed that acrolein induced alterations in protein secondary structure of the porcine SC sheets, which were accompanied by diminished fibrous keratin structure observed by transmission electron microscopy. These results show the possibility that carbonylation of the SC caused by environmental factors is one of factors altering the fibrous structure of keratin and decreasing the light transmission of SC, which changes the quality of the skin appearance.     

Seasonal influences on stratum corneum ceramide 1 fatty acids and the influence of topical essential fatty acids

Ceramide 1 is the main repository of stratum corneum linoleic acid, and changes in the levels of ceramide 1 linoleate are associated with cutaneous abnormalities: essential fatty acid deficiency, atopic dermatitis and acne. We have previously reported seasonal variation in stratum corneum lipids where lipids, particularly ceramides, are very much reduced in winter which probably influences the appearance of skin xerosis in this season. However, investigations into the seasonal variation in ceramide 1 fatty acid levels and the effects of topical essential fatty acids on the composition of these lipid species have not been conducted in healthy subjects. We determined the composition of stratum corneum ceramide 1 esterified fatty acids in leg skin from Caucasian women in winter and summer and also investigated the effects of topical triglycerides rich in linoleic acid on stratum corneum ceramide 1 fatty acid levels in winter. A dramatic seasonal variation was observed. Whereas decreased levels of linoleic acid and some saturated fatty acids (C-15 and C-24) were observed in the winter months, increased levels of oleic acid were found. Nevertheless, it was possible to normalize the levels of stratum corneum ceramide 1 linoleate following topical application of formulations containing linoleic acid-rich triglycerides for 4 weeks. The reduction in ceramide 1 linoleate may lead to impaired stratum corneum function and increased susceptibility to skin xerosis in winter. However, by using formulations containing linoleic acid esters it is possible to correct these changes and possibly improve barrier resilience.     

Evolution of lipid composition in skin treated with blackcurrant seed oil

This report utilizes a new procedure based on epidermal stripping to analyse unlabelled skin fatty acids. This technique was applied to the study of human dry skin treated with blackcurrant seed oil, an essential fatty acid rich lipid also containing γ linolenic acid.
In human dry skin the usual fatty acids were found in the stratum corneum as in normal skin outer layers. Among the saturated series no significant difference was observed in the C16 chain but a smaller quantity of C18:0 was detected. In the unsaturated series a marked fall of linoleic acid as well as a higher level of C16:1 in the stratum corneum appeared as the major significant events that could characterize dry skin. γ linolenic acid was detectable neither in normal nor in dry skin. α linolenic acid was found but not consistently.
After multiple applications of blackcurrant seed oil in an emulsion preparation, impregnation of the stratum corneum was complete, and essential or other fatty acids contained in the oil (particularly linoleic and linolenic acids) were found in combination with endogenous lipids. Possibly they contribute to enhance the protective effect of the stratum corneum.     

Methodology for Evaluation of Skin Transparency and the Efficacy of an Essence That Can Improve Skin Transparency

Transparency of the skin is an important factor for realizing beautiful skin, but there is only a visual sensory evaluation method by a beauty specialist. It is unclear how skin physiological characteristics can give transparency. Our purpose is to establish an objective method for evaluating the transparency of the skin and to develop a special cosmetics product which can enhance skin transparency, after investigating the mechanism of the skin characteristics which yield transparency. We developed an optical instrument that could independently measure a diffuse reflection as well as a specular reflection by assembling a polarizing filter in a gonio-spectrum photometer. We analyzed the relationship among skin transparency, specular reflection light and diffuse reflection light. It was shown that there was a strong positive correlation between the skin transparency and the intensity of the diffuse reflection light ( r  = 0.699) and no correlation between the skin transparency and the intensity of the specular reflection light ( r  = 0.190). The results of partial least squares (PLS) analysis of the skin transparency and the skin physiological characteristics showed that a highly transparent skin has a fine skin surface texture with a high hydration of stratum corneum, a low melanin content and a low hemoglobin content. On the basis of these results, we developed an essence that can improve skin texture and blood flow, reduce melanin content, and increase the moisture in the stratum corneum. After eight weeks' usage of this product by consumer panels ( n  = 30), it was confirmed that the skin transparency was improved by the increase of the intensity of the diffuse reflection light by gonio-spectrum photometer measurement and by consumers' self-assessment.     

Stratum corneum keratin structure, function and formation - a comprehensive review

A comprehensive review on stratum corneum keratin organization, largely based on the recently published cubic rod-packing and membrane templating model [J. Invest. Dermatol., 123, 2004, 715], is presented. Keratin is the major non-aqueous component (wt/wt) of stratum corneum. As 90-100% of the stratum corneum water is thought to be located intracellularly one may presume that keratin also is a major factor (together with filaggrin-derived free amino acids) determining stratum corneum hydration level and water holding capacity. This water holding capacity depends in turn on the structural organization of the corneocyte keratin intermediate filament network. The cubic rod-packing model for the structure and function of the stratum corneum cell matrix postulates that corneocyte keratin filaments are arranged according to a cubic-like rod-packing symmetry. It is in accordance with the cryo-electron density pattern of the native corneocyte keratin matrix and could account for the swelling behaviour and the mechanical properties of mammalian stratum corneum. The membrane templating model for keratin dynamics and for the formation of the stratum corneum cell matrix postulates the presence in viable epidermal cellular space of a highly dynamic small lattice parameter (<30 nm) membrane structure with cubic-like symmetry, to which keratin is associated. It further proposes that membrane templating, rather than spontaneous self-assembly, is responsible for keratin intermediate filament formation and dynamics. It is in accordance with the cryo-electron density patterns of the native keratinocyte cytoplasmic space and could account for the characteristic features of the keratin network formation process, the dynamic properties of keratin intermediate filaments, the close lipid association of keratin, the insolubility in non-denaturating buffers and pronounced polymorphism of keratin assembled in vitro, and the measured reduction in cell-volume and hydration level between stratum granulosum and stratum corneum.     

Skin barrier structure, function and formation - learning from cryo-electron microscopy of vitreous, fully hydrated native human epidermis

The hydration level of the stratum corneum largely depends on the physical state and molecular organization of the stratum corneum intercellular lipid matrix. A better understanding of stratum corneum lipid organization may thus aid the development of more rational cosmetic formulations. Several experimental and theoretical problems of a fundamental character remain, however, unresolved. These are, e.g. that precise quantitative skin barrier compositional data are difficult to obtain and that in vitro experimental skin barrier models usually are of limited value because of the prevailing non-equilibrium conditions in vivo. However, new experimental methods have recently been developed, which may help to overcome some of these limitations. These are, e.g. direct electrospray ionization-mass spectrometry (ESI-MS) of intact long-chain skin ceramides and direct high-resolution cryo-transmission electron microscopy (cryo-TEM) of vitreous sections of native, fully hydrated epidermis. Here, we show that cryo-transmission electron micrographs of vitreous normal human skin often dramatically differ from those obtained by conventional electron microscopy of resin-embedded skin. Our epidermal cryo-TEM data are subsequently discussed in relation to central problems of present conceptions of skin barrier structure, function and formation.     

Skin lipid organization,composition and barrier function

Citation: IFSCC Magazine, 10 (2007) (4) 297–307 The primary function of the skin is to act as a barrier against unwanted influences from the environment and to protect the body from water loss. The barrier function of the skin is located in the superficial layer of the skin, the stratum corneum. The stratum corneum consists of dead cells filled with keratin and water, the corneocytes, embedded in lipid regions. As the lipid regions are the only continuous structure in the stratum corneum, they are considered to be very important for the skin barrier function. The main lipid classes are ceramides, cholesterol and free fatty acids. In this paper the lipid organization in human stratum corneum is reviewed. In addition, the role the various lipid classes play in lipid organization is discussed using mixtures prepared from either native human ceramides or synthetic ceramides. Finally, a model is described which allows study of the relation between lipid composition, organization and barrier function. This model is referred to as the stratum corneum substitute. Keywords: ceramides, FTIR, lipid organization, stratum corneum, X‐ray diffraction     

J. Cosmet. Sci., 59, 59–69 (January/February 2008)Biological activities of selected peptides: Skin penetration ability of copper complexes with peptides

This study concerning the permeability through skin barriers of copper complexes with peptides is an important part of the research on their biological activity. The transport of copper complexes through the skin is essential in treatment of dermatological dysfunctions connected to the deficiency of these elements in the skin. During the last several years, a special interest in transepidermal copper delivery has been observed. This is the reason why copper compounds have been used as active compounds in care cosmetics. Yet, the transport process of copper complexes with tripeptides, glycyl-histidyl-lysine GHK, or γ-glutamylcysteinyl-glycine GSH through the stratum corneum has received very little attention in the literature so far. The penetration ability of GHK-Cu and GSH-Cu through the stratum corneum and the influence of the complexes with tripeptide on the copper ion transport process is the key factor in their cosmetic and pharmaceutical activity. The in vitro penetration process was studied in the model system, a Franz diffusion cell with a liposome membrane, where liquid crystalline systems of physicochemical properties similar to the ones of the intercellular cement of stratum corneum were used as a standard model of a skin barrier. The results obtained demonstrated that copper complexes permeate through the membranes modeling the horny lipid layer and showed the influence of peptides on the dynamics of copper ion diffusion.     

Trends in stratum corneum research and the management of dry skin conditions

The structure, composition, formation and function of the stratum corneum have been the subject of intense research over the last few decades. As has become apparent, stratum corneum barrier function is not only dependent on one single component but also on its total architecture. Recent developments in understanding lipid composition have led to a new ceramide nomenclature system, a new proposal for a molecular model of the interactions between ceramides, cholesterol and fatty acids, and the demonstration of the presence of crystalline orthorhombic and gel hexagonal lipid phases in the stratum corneum. Linoleate-containing ceramide one, now known as CER EOS, have been shown to be essential for the formation of the 13 nm long periodicity phase (LPP) observed by electron microscopy and X-ray diffraction studies, whereas long-chain fatty acids are important for the formation of the crystalline lipid phases essential for barrier function. The role of the corneocyte envelope, its constituent proteins and its transglutaminase-mediated maturation processes have been shown to be essential for good skin condition. Several proteases may have a role in corneodesmolysis, particularly serine and cathepsin-like enzymes. Novel filaggrin polymorphisms have been identified that may be involved in the expression of a dry skin phenotype. Disturbances in lipid packing states, reduction in ceramide levels (particularly the phytosphingosine-containing ceramides), reductions in the levels of long-chain fatty acids and loss of the LPP largely account for the perturbations in lipid structure that occur in dry skin. The reduced corneodesmolysis that occurs in this xerotic skin disorder is now well accepted and is caused by reductions in the levels and activities of stratum corneum proteases together with elevated levels of corneodesmosomal glycoproteins in the superficial layers of the stratum corneum. Additionally, increased levels of fragile corneocytes are associated with reduced transglutaminase activity and corneocyte envelope cross-linking events. However, in comparison with the advances in our understanding of the textural changes that occur in dry skin, the somatosensory changes are poorly understood and the itching associated with dry skin is still an under-researched area. The unique biosensor role of the stratum corneum essential for a competent natural moisturizing barrier may also have a role to play in the action of anti-ageing technologies by controlling the expression and secretion of epidermal cytokines and growth factors. Technologies to treat the surface textural skin problems, enhance the differentiation process, particularly lipid biosynthesis, and to control the somatosensory problems in dry skin have received much attention in the last decade. This paper will review the state of the art of stratum corneum biology and the trends in the management of dry skin.     

Improvement in skin color achieved by smoking cessation

It has been well known that habitual smoking accelerates premature skin ageing recognized as ‘smoker's face’. However, the effect of smoking cessation on the appearance of skin has not been elucidated. The aim of this study was to evaluate objectively the effect of smoking cessation on the skin's appearance. The stratum corneum carbonyl protein level and skin colour of the cheek and the hand were measured. The change before and during the smoking cessation treatment (0, 2, 4, 8 and 12 weeks), and the success or failure in smoking cessation, was compared and examined. Eighty‐four cases who had smoking cessation treatment were examined. The level of the stratum corneum carbonyl protein did not show any difference comparing before and after treatment for the smoking cessation success group and the failure group. The lightness of skin colour showed an upward tendency 4–12 weeks after starting the treatment in the success group and increased significantly compared with the failure group. The redness showed a significant decrease in comparison with before the treatment, and it also showed a significant decrease compared with the failure group. The yellowness did not show any clear tendency. Also, the haemoglobin showed a decreased tendency. Furthermore, multivariate statistical analysis showed a possibility that the lightness and haemoglobin could be changed by smoking cessation treatment. In conclusion, our study showed that an upward tendency of skin lightness was seen to correspond with a haemoglobin decrease accompanied by smoking cessation. If we can easily measure skin improvement as an effect of smoking cessation, it is thought to be a useful aid for smoking cessation support.     

Increased carbonyl protein levels in the stratum corneum of the face during winter

The stratum corneum (SC) is the interface between the body and the environment, and is continuously exposed to oxidative stress that results in carbonyl modification of proteins. We previously developed a simple and non-invasive method to assess the stratum corneum carbonyl protein (SCCP) levels. In this study, we used this method to examine the seasonal changes in the SCCP levels and the relationship between the SCCP level and the physiological condition of the SC. SC was collected from the face of healthy Japanese volunteers by adhesive tape stripping and its carbonyl groups were determined by reaction with fluorescein-5-thiosemicarbazide. The average fluorescence intensity of the SC was calculated as the SCCP level. The SCCP level in the cheek was higher in winter than summer. The SCCP level was negatively correlated with the water content in the SC measured by the conductance and capacitance, and also negatively correlated with the extensibility of the skin measured by a Cutometer, suggesting that the mechanical properties of the skin can be affected by oxidative modification of the SC. These data suggest the involvement of oxidative modification of SC proteins in the generation of rough skin during winter.     

Human skin equivalents are an excellent tool to study the effect of moisturizers on the water distribution in the stratum corneum

In this study, the mode of action of moisturizers on the level of water in the stratum corneum was studied using cryo‐scanning electron microscopy. As model for dry skin, we used human skin equivalents (HSEs) generated at 93% or 60% relative humidity (RH). During the generation of the HSEs, the moisturizers were applied during a period of maximal 2 weeks. In HSEs generated under normal culture conditions (93% RH), application of 10% glycerol or 5% urea formulations resulted in increased water levels. Whereas the 5% urea formulations resulted mainly in the formation of intercellular water domains, after 10% glycerol both swelling of corneocytes and formation of intercellular water domains were noticed. A reduction in RH to 60% during treatment reduced the stratum corneum water levels drastically. Treatment with the non‐occlusive lipophilic moisturizer isopropyl isostearate resulted in increased water level in the central part of the stratum corneum compared with the untreated control. Our results show that HSEs can be used as a model to study the water distribution.     

Investigation of interactions between silicones and stratum corneum lipids

Ingredients of topically applied skin care formulations have not only positive effects on the appearance of human skin but can also disturb the Stratum corneum (SC) lipid barrier. In the present study, the influence of silicones (PDMS), as often used cosmetic ingredients, on the microstructure of SC lipids was investigated. For this purpose the interactions of four different PDMS with excised human SC were examined first using differential scanning calorimetry (DSC) and wide angle X-ray diffraction for physical characterization. Because the physical properties of human stratum corneum strongly depend on the lipid composition, showing inter-and intra-individual differences, the interactions with an in vitro model lipid system containing SC fatty acids were also studied, using polarized light microscopy, transmission electron microscopy, small angle X-ray diffraction and DSC. The results revealed that the investigated PDMS do not change either the microstructure of excised human SC or the biphasic lamellar/inverse hexagonal structure of the in vitro model. We concluded that PDMS will not cause any side-effects when topically applied and that our simplified in vitro model could be helpful for estimating interactions between cosmetic ingredients and other topically applied substances and the skin barrier at an early moment of formulation development.     

Infrared spectroscopy of the skin: influencing the stratum corneum with cosmetic products

The stratum corneum is the outermost layer of the skin and, as such, represents the decisive barrier between the body and the environment. The combination of horny cells and lamellar lipid layers prevents water loss from the body and protects it against penetration by exogenous substances. For selective investigation of the thin outermost stratum corneum layer ATR-FTIR (attenuated total reflection Fourier transform infrared) spectroscopy has proved itself in practice. It provides information about the sebum content, type of fatty substances, water content and degree of order of the lamellar lipid film in the stratum corneum. Different types of skin (dry, normal and greasy) differ mainly in their sebum content but also in the composition of their fats and the degree of order of their lipids. A comparison with model lipid mixtures shows that the stratum corneum lipids are mainly present in a fixed gel form under physiological conditions. In cleansing processes a large part of the sebum is removed from the skin. The skin reacts to the defatting process by the rapid secretion of sebum. After cleansing with a mild face cleansing emulsion the re-achievement of the same fat status takes longer than with a surfactant shower gel. Skin creams cause alterations to the skin moisture and in the degree of order of the stratum corneum lipids. A lamellar cream with a structure similar to that of the stratum corneum lipids increases the degree of order of the alkyl chains of the skin lipid film (biomimetic principle), whereas a conventional w/o cream reduces this degree of order. Skin moisture increases after the use of the creams.     

A new technique for the evaluation of cosmetics effect on mechanical properties of stratum corneum and epidermis in vitro

The mechanical properties of stratum corneum and epidermis have been measured in vitro and a dispersity of approximately 30% to 50% between samples from the same donor has been found. To overcome such a limitation, a technique was developed in order to compare the mechanical properties of the same skin sample before and after treatment. The chosen parameter (initial slope of the stress-strain curve) appeared to be influenced by the topical application of products. A different time response was found with stratum corneum and epidermis reflecting their structural differences.
Une nouvelle technique d'evaluation de l'effet des produits cosmétique sur les propriétés mécaniques du stratum corneum et de l'épiderme humains (etude in vitro )     

Eucalyptus increases ceramide levels in keratinocytes and improves stratum corneum function

The objectives of this study were to identify a plant extract that would improve stratum corneum functions and to elucidate the mechanism(s) involved. Based on the information that stratum corneum functions depend on the level of ceramide in the stratum corneum, we identified a Eucalyptus extract that was able to increase the level of ceramide in human keratinocytes in culture and in human stratum corneum and that improves the stratum corneum water holding and barrier functions. Addition of the Eucalyptus extract to human keratinocytes in culture increased the level of ceramide in a dose-dependent manner and also increased the biosynthesis of ceramide, glucosylceramide and sphingomyelin. Topical application of the Eucalyptus extract on the dry skin of human subjects induced by acetone and diethylether treatment resulted in a significant increase in ceramide level in the stratum corneum, a significant improvement in its water-holding function and an improvement in its barrier function. The addition of macrocarpal A, one of the main components of the Eucalyptus extract, to human keratinocytes in culture increased the level of ceramide and the mRNA expression of serine palmitoyltransferase, acid sphingomyelinase, neutral sphingomyelinase, glucosylceramide synthase and glucocerebrosidase in a dose-dependent manner. Our results indicate that the increased content of ceramides in the stratum corneum may underlie the therapeutic effect of the Eucalyptus extract. Our results also indicate the possibility that macrocarpal A is the key component that stimulates the synthesis of ceramide in the stratum corneum.     

In vivo methods for the analysis of the penetration of topically applied substances in and through the skin barrier

The efficacy of a drug is characterized by its action mechanism and its ability to pass the skin barrier. In this article, different methods are discussed, which permit this penetration process to be analysed non‐invasively. Providing qualitative and quantitative information, tape stripping is one of the oldest procedures for penetration studies. Although single cell layers of corneocytes are removed from the skin surface, this procedure is considered as non‐invasive and is applicable exclusively to the stratum corneum. Recently, optical and spectroscopic methods have been used to investigate the penetration process. Fluorescence‐labelled drugs can be easily detected in the skin by laser scanning microscopy. This method has the disadvantage that the dye labelling changes the molecular structures of the drug and consequently might influence the penetration properties. The penetration process of non‐fluorescent substances can be analysed by Raman spectroscopy, electron paramagnetic resonance, CARS and multiphoton microscopic measurements. Using these methods, the concentration of the topically applied formulations in different depths of the stratum corneum can be detected by moving the laser focus from the skin surface deeper into the stratum corneum. The advantages and disadvantages of these methods will be discussed in this article.     

The ultraviolet microscopic transmission characteristics of human stratum corneum

This study has evaluated the ultraviolet light transmission characteristics of human stratum corneum at the single cell level using a low light level video microscope to measure the mean percentage transmission of light at different wavelengths and the variation in transmission across the stratum corneum. Stratum corneum was isolated by an enzymic technique and examined on a low light level UV video microscope. Quantitative evaluation of the transmitted monochromatic light for the underside of the layer was measured directly using a Kontron UNIPS image processor or indirectly with a Quantimet 920 image processor after video recording. Transmission distribution histograms were obtained from samples of stratum corneum taken from human breast, scalp, abdomen and leg. Mean transmission values were also derived and compared with diffuse transmission values obtained using the same tissue mounted on an integrating sphere. The UV microscopic transmission characteristics of enzyme separated stratum corneum clearly demonstrated that this structure was not an ideal diffuser. Uniform light intensity on the surface of the stratum corneum led to areas of transmitted intensity in close proximity that differed by factors ofthree to six fold, e.g. between regions of high (>70%) and low (<20%) brightness. However, the average transmission was found to be compatible with published data obtained by diffuse transmission spectrophotometry, taking into account the enhanced transmission arising from stratum corneum immersion in phosphate buffered saline. This was confirmed by the elevated values obtained by diffuse transmission spectrophotometry in this study for samples of stratum corneum prepared for UV microscopy being higher than these found in published data. It is obvious from these findings that viable cells in the epidermis are not exposed to a uniform incident light intensity even when this is true for the surface of skin. Studies of skin response to ultraviolet light at the single cell level must take account of the possibility of preferential exposure of specific sites in any subsequent explanation of cell sensitivity. This is in addition to the already well established cell cycle dependent ultraviolet sensitivity.