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Understanding the changes in the skin microbiome and their relationship to host skin factors during aging remains largely unknown. To better understand this phenomenon, we collected samples for metagenomic and host skin factor analyses from the forearm, buttock, and facial skin from 158 Caucasian females aged 20‒24, 30‒34, 40‒44, 50‒54, 60‒64, and 70‒74 years. Metagenomics analysis was performed using 16S ribosomal RNA gene sequencing, whereas host sebocyte gland area, skin lipids, natural moisturizing factors, and antimicrobial peptides measurements were also performed. These analyses showed that skin bacterial diversity increased at all the skin sites with increasing age. Of the bacterial genera with an average relative abundance1%, only Lactobacillus and Cutibacterium demonstrated a significant change (decrease) in abundance at all sampled skin sites with increasing age. Additional bacterial genera demonstrated significant age- and site-specific changes in abundance. Analysis of sebocyte area, natural moisturizing factors, lipids, and antimicrobial peptides showed an age-related decrease in sebocyte area and increases in natural moisturizing factors/antimicrobial peptides/skin lipids, all of which correlated with changes in specific bacterial genera. In conclusion, the human skin microbiome undergoes age-associated alterations that may reflect underlying age-related changes in cutaneous biology.

Individuals suffering from sensitive skin often have other skin conditions and/or diseases, such as fair skin, freckles, rosacea, or atopic dermatitis. Genome-wide association studies (GWAS) have been performed for some of these conditions, but not for sensitive skin. In this study, a total of 23,426 unrelated participants of European ancestry from the 23andMe database were evaluated for self-declared sensitive skin, other skin conditions, and diseases using an online questionnaire format. Responders were separated into two groups: those who declared they had sensitive skin (n = 8971) and those who declared their skin was not sensitive (controls, n = 14,455). A GWAS of sensitive skin individuals identified three genome-wide significance loci (p-value < 5 × 10−8) and seven suggestive loci (p-value < 1 × 10−6). Of the three most significant loci, all have been associated with pigmentation and two have been associated with acne.

Background Intrinsic and extrinsic factors, including ultraviolet irradiation, lead to visible signs of skin aging. Objective We evaluated molecular changes occurring in photoexposed and photoprotected skin of white women 20 to 74 years of age, some of whom appeared substantially younger than their chronologic age. Methods Histologic and transcriptomics profiling were conducted on skin biopsy samples of photoexposed (face and dorsal forearm) or photoprotected (buttocks) body sites from 158 women. 23andMe genotyping determined genetic ancestry. Results Gene expression and ontologic analysis revealed progressive changes from the 20s to the 70s in pathways related to oxidative stress, energy metabolism, senescence, and epidermal barrier; these changes were accelerated in the 60s and 70s. The gene expression patterns from the subset of women who were younger-appearing were similar to those in women who were actually younger. Limitations Broader application of these findings (eg, across races and Fitzpatrick skin types) will require further studies. Conclusions This study demonstrates a wide range of molecular processes in skin affected by aging, providing relevant targets for improving the condition of aging skin at different life stages and defining a molecular pattern of epidermal gene expression in women who appear younger than their chronologic age.

In human skin fibroblasts in vitro, procollagen-1 and NAD(+)/NADH were reduced in three strains of adult fibroblasts compared with neonatal fibroblasts. The levels of both procollagen-1 and NAD(+)/NADH were increased in the adult fibroblasts by treatment for 24 (NAD energy) or 48 h (procollagen-1) with a complex containing niacinamide, Pal-KTTKS peptide and an olive oil fatty acid derivative (Olivem(®)), especially in combination with a natural extract from dill (Lys'lastine V(®)). In one of the adult fibroblast strains evaluated, these changes in procollagen-1 and NAD(+)/NADH in response to the complex of bioactives were in parallel with increased expression of mRNA biomarkers related primarily to dermal matrix and basement membrane structure, including COL1A1, COL3A1, COL5A1, COL14A1, ELN and LOXL2, in addition to SOD2, NAMPT and TGFBR3; MMP1 was decreased in expression. In general, these mRNA biomarker effects were maintained or boosted by the addition of Lys'lastine V, particularly at 1%, and were similar to the fold changes in mRNA expression in neonatal compared with adult fibroblasts. These results indicate that the complex of niacinamide, Pal-KTTKS and Olivem, especially with addition of Lys'lastine V, increases the NAD(+)/NADH bioenergy level of adult skin fibroblasts in parallel with increased expression of skin structure biomarkers in vitro to levels similar to those in younger fibroblasts. Thus, niacinamide, Pal-KTTKS, Olivem and Lys'lastine V are promising bioactive candidates for inclusion in cosmetic formulations.

The use of global gene expression profiling, also known as transcriptomics or genomics, provides a means to identify key pathways affected in ageing skin that can be improved with appropriate cosmetic compounds. Aspects of skin ageing that can be addressed include matrix production, barrier, lipid synthesis, antioxidant capacity and hyperpigmentation. Gene expression profiling together with in vitro human skin cell cultures for compound screening and verification have led to the identification of cosmetic compounds and an understanding of the biological effects of compounds such as niacinamide, Pal-KTTKS, hexamidine, retinyl propionate and sodium dehydroacetate. In addition, understanding of the decreased antioxidant capacity of aged skin has led to the identification of new antiageing ingredients, olive-derived fatty acid ethoxylates, which have been shown to restore antioxidant enzymes in skin keratinocytes and fibroblasts. Gene expression profiling of age spots has also provided an understanding of the role of undecylenoyl phenylalanine in reducing melanin production by an adrenergic receptor mechanism in melanocytes. The use of these compounds in cosmetic formulations for skin care can aid improvements in the appearance of aged skin, including the improved appearance of fine lines, wrinkles and age spots.

The skin has an amazing array of complex interacting biological processes. Recent advances in investigational techniques now allow evaluation of these processes at the level of the gene, protein and metabolite. Sometimes collectively known as the omics, these fields of inquiry, known as genomics, proteomics and metabolomics, respectively, are yielding new and important insights into skin structure and processes, its responses to injury and age, and the mechanisms by which new interventions and compounds may work to improve the health and integrity of this crucial organ.

Prior to the manufacture, transport, and marketing of chemicals or products, it is critical to assess their potential for skin toxicity (corrosion or irritation), thereby protecting the worker and consumer from adverse skin effects due to intended or accidental skin exposure. Traditionally, animal testing procedures have provided the data needed to assess the more severe forms of skin toxicity, and current regulations may require animal test data before permission can be obtained to manufacture, transport, or market chemicals or the products that contain them. In recent years, the use of animals to assess skin safety has been opposed by some as inhumane and unnecessary. The conflicting needs of the industrial toxicologist to (1) protect human safety, (2) comply with regulations, and (3) reduce animal testing have led to major efforts to develop alternative, yet predictive, test methods. A variety of in vitro skin corrosion test methods have been developed and several have successfully passed initial international validation. These have included skin or epidermal equivalent assays that have been shown to distinguish corrosive from noncorrosive chemicals. These skin/epidermal equivalent assays have also been modified and used to assess skin irritation potential relative to existing human exposure test data. The data show a good correlation between in vitro assay data and different types of human skin irritation data for both chemicals and consumer products. The effort to eliminate animal tests has also led to the development of a novel human patch test for assessment of acute skin irritation potential. A case study shows the benefits of in vitro and human skin irritation tests compared to the animal tests they seek to replace, and strategies now exist to adequately assess human skin irritation potential without the need to rely on animal test methods.

Eye irritation potency of a compound or mixture has traditionally been evaluated using the Draize rabbit-eye test (Draize et al., 1944). In order to aid predictions of eye irritation and to explore possible corresponding mechanisms of eye irritation, a methodology termed "membrane-interaction QSAR analysis" (MI-QSAR) has been developed (Kulkarni and Hopfinger 1999). A set of Draize eye-irritation data established by the European Center for Ecotoxicology and Toxicology of Chemicals (ECETOC) (Bagley et al., 1992) was used as a structurally diverse training set in an MI-QSAR analysis. Significant QSAR models were constructed based primarily upon aqueous solvation-free energy of the solute and the strength of solute binding to a model phospholipid (DMPC) monolayer. The results demonstrate that inclusion of parameters to model membrane interactions of potentially irritating chemicals provides significantly better predictions of eye irritation for structurally diverse compounds than does modeling based solely on physiochemical properties of chemicals. The specific MI-QSAR models reported here are, in fact, close to the upper limit in both significance and robustness that can be expected for the variability inherent to the eye-irritation scores of the ECETOC training set. The MI-QSAR models can be used with high reliability to classify compounds of low- and high-predicted eye irritation scores. Thus, the models offer the opportunity to reduce animal testing for compounds predicted to fall into these two extreme eye-irritation score sets. The MI-QSAR paradigm may also be applicable to other toxicological endpoints, such as skin irritation, where interactions with cellular membranes are likely.

Human corneal equivalents comprising the three main layers of the cornea (epithelium, stroma, and endothelium) were constructed. Each cellular layer was fabricated from immortalized human corneal cells that were screened for use on the basis of morphological, biochemical, and electrophysiological similarity to their natural counterparts. The resulting corneal equivalents mimicked human corneas in key physical and physiological functions, including morphology, biochemical marker expression, transparency, ion and fluid transport, and gene expression. Morphological and functional equivalents to human corneas that can be produced in vitro have immediate applications in toxicity and drug efficacy testing, and form the basis for future development of implantable tissues.

The appearance of corneal opacity in a rabbit eye irritation test is of concern due to the importance of corneal clarity to vision. An important consideration, both for human safety assessments and regulatory classifications, is whether the corneal opacity is readily and completely reversible, or is irreversible. In fact, new harmonized regulatory guidelines for classification of eye irritants and corrosives assume that the most severe of corneal opacities (grade 4) are irreversible. The purpose of the current work was to use an extensive historical database to examine this assumption and to identify factors that are important in reversibility. Eye test scores from historic rabbit eye irritation tests were analyzed to determine the reversibility within 21 days of corneal opacity responses graded as 4, on a scale of 0-4, at 1, 2, or 3 days after instillation of test material, and to define factors influencing reversibility. Overall, the historic data indicated that 45% of corneal opacities graded a...

Cell function-based tests measure responses of cells at sublytic concentrations of test agents. The fluorescein leakage assay measures effects of substances on the barrier function of epithelial monolayers or multilayers (MDCK or NHEK cells) as in vitro models of corneal epithelial function. Two IRAG data submissions suggest that the fluorescein leakage assay shows promise as a screening test for surfactants and alcohols. The test method requires further optimization, standardization and evaluation to fully determine its utility as an in vitro ocular irritancy test. The silicon microphysiometer test measures effects of test substances on the metabolic rate of cells; although a large number of cell types have been evaluated, L929 cells have been used for irritancy screening. Three IRAG data submissions on the silicon microphysiometer test showed strong in vivo/in vitro correlations for surfactants and surfactant-based personal care and household cleaning products in a range of mild to moderate ocular irritancy. This and published information support the reproducibility of the method and its use as an ocular irritancy screening test for aqueous-soluble liquid, surfactant-based formulations.

As part of the Interagency Regulatory Alternatives Group (IRAG) program to evaluate the state of the art in the development of alternative (non-whole animal) eye irritation tests, academic and industrial organizations were invited to submit in vitro eye irritation data generated in their laboratories to one of several working groups for review. The assays reviewed in this report (from Working Group 5, “Other Assays”) were the EYTEX™ assay, tissue equivalent assay, a cytotoxicity assay using three-dimensional human fibroblast constructs, the Microtox™ assay, and other miscellaneous assays. Each submission consisted of raw data for chemicals and products tested, a description of the methodology, and an analysis (generally by regression analysis and Pearson's correlation coefficient) for the performance of the in vitro test relative to its ability to predict individual ocular tissue scores or total ocular score. In vivo data were generated according to the scoring methods proposed by Draize. Working Group 5 evaluated the submissions and commented on the utility of the assays. The variability of the in vivo data made conclusions difficult in many situations. Most of these assays were deemed useful (within limited chemical classes) for screening purposes or for use in conjunction with other toxicological information.

The self-assembly of three cosmetically active peptide amphiphiles C16-GHK, C16-KT, and C16-KTTKS (C16 denotes a hexadecyl, palmitoyl chain) used in commercial skin care products is examined. A range of spectroscopic, microscopic, and X-ray scattering methods is used to probe the secondary structure, aggregate morphology, and the nanostructure. Peptide amphiphile (PA) C16-KTTKS forms flat tapes and extended fibrillar structures with high β-sheet content. In contrast, C16-KT and C16-GHK exhibit crystal-like aggregates with, in the case of the latter PA, lower β-sheet content. All three PA samples show spacings from bilayer structures in small-angle X-ray scattering profiles, and all three have similar critical aggregation concentrations, this being governed by the lipid chain length. However, only C16-KTTKS is stained by Congo red, a diagnostic dye used to detect amyloid formation, and this PA also shows a highly aligned cross-β X-ray diffraction pattern consistent with the high β-sheet content in the self-assembled aggregates. These findings may provide important insights relevant to the role of self-assembled aggregates on the reported collagen-stimulating properties of these PAs.

National and international regulations require that chemicals must be properly classified, labeled, packaged, and transported based on their ability to damage or destroy tissue, e.g., skin. Traditionally, skin corrosion assessments were based on tests involving topical application of test substances to the skin of rabbits. In the present work, anin vitroskin corrosion test based on the use of reconstructed human skin cultures was developed as a potential replacement forin vivorabbit skin tests for corrosion. In thein vitromethod, test substances were applied topically to the stratum corneum surface of human skin cultures. Skin culture damage or cytotoxicity was measured as decreased 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide (MTT) vital dye metabolism. In time-course experiments, the time (in minutes) of test material exposure eliciting a 50% reduction of MTT metabolism (i.e.,t50 value) was calculated. Using this method we evaluated 24 chemicals and found that the 9 corrosive chemicals were accurately distinguished from 15 strong, moderate, or mild skin irritants, using anin vitrocutoff oft50

The reproducibility of toxicologic test methods, including alternative tests, is a key scientific and regulatory concern. In the present work, historic rabbit eye irritation data were used to determine the intra- and interlaboratory reproducibility of the low volume eye test (LVET). The standard Draize eye irritation test was used as the basis for comparison. The LVET and Draize tests had similar degrees of intra- and interlaboratory reproducibility as determined by examination of their coefficients of variation, although the variability in LVET results was directionally lower. Results from 70 parallel Draize and LVET tests indicated a strong positive association between results from the two tests, for corneal, iridial, conjunctival, and maximum average scores (MAS). Correlation coefficients were 0.60, 0.73, 0.69, and 0.73, respectively ( P ≤ 0.0001). The linear relationship between LVET and Draize MAS values was examined by regression analysis and found to follow the relationship LVET MAS = 0.522 (Draize MAS). Thus, the LVET is at least as reproducible as the Draize test and gives responses that are (linearly) correlated to the Draize. The previous findings that the LVET is more predictive of human eye responses than the Draize test lends additional support for its use as a refined alternative to the Draize test.

National and international regulations require that chemicals must be properly classified, labeled, packaged, and transported based on their ability to damage or destroy tissue, e.g., skin. Traditionally, skin corrosion assessments were based on tests involving topical application of test substances to the skin of rabbits. In the present work, an in vitro skin corrosion test based on the use of reconstructed human skin cultures was developed as a potential replacement for in vivo rabbit skin tests for corrosion. In the in vitro method, test substances were applied topically to the stratum corneum surface of human skin cultures. Skin culture damage or cytotoxicity was measured as decreased 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide (MTT) vital dye metabolism. In time-course experiments, the time (in minutes) of test material exposure eliciting a 50% reduction of MTT metabolism (i.e., t50 value) was calculated. Using this method we evaluated 24 chemicals and found that the 9 corrosive chemicals were accurately distinguished from 15 strong, moderate, or mild skin irritants, using an in vitro cutoff of t50 < 3 min. Histologic examination of the cultures indicated gradations of epidermal necrosis quantitated using a specially designed grading scale, which correlated well with the corrosivity of treatment chemicals and cytotoxicity measurements. The predictivity of the method was confirmed and was consistent in skin culture models from two suppliers. Thus, the utility of human skin equivalent cultures as a screening tool for prediction of skin corrosivity appears to be independent of the commercial source of the cultures. We conclude that the in vitro assay using human skin equivalent cultures is a promising alternative to in vivo rabbit skin corrosion tests for assessment of the corrosivity of chemicals to human skin.

Cultured human skin cells are a potentially useful model for skin irritancy testing. We have evaluated the effects of chemical irritants on human epidermal keratinocytes (NHEK) and on keratinocyte-dermal fibroblast (NHEK/DF) co-cultures. Cell viability in NHEK cultures, measured as incorporation of the vital dye neutral red (NR), was reduced in a dose-dependent manner in response to the chemical irritants tested. The half-maximal effective concentration (NR(50)) values correlated with irritation scores in human patch tests with these materials. Certain materials were found to be incompatible with this test system. NHEK/DF cultures were treated with ten prototype surfactants, and were evaluated for cell viability (MTT incorporation), cytotoxicity (release of the enzymes lactate dehydrogenase and N-acetyl glucosaminidase), metabolism (glucose utilization), and inflammatory mediator (prostaglandin E(2)) release. There was a close correlation of the dose-response characteristics for all the endpoints tested, and between the in vitro responses and human patch test scores for the surfactants tested. These results demonstrate the usefulness of human skin cell cultures and of cell viability, cytotoxicity, and inflammatory mediator release as endpoints, for the in vitro assessment of skin irritancy.

The tissue equivalent assay (TEA) (Osborne et al., 1995) was used to evaluate 55 mixed ingredients and formulations in the COLIPA International Validation Study on Alternatives to the Draize Rabbit Eye Irritation Test (Brantom et al., 1997). The TEA can be used to test all types of materials since it uses a topical application approach and is not limited to only testing liquid or soluble materials. A prediction model (PM) for the test was developed using historical eye irritation data from a total of 132 materials on which in vivo and in vitro data were available. A regression model was derived from these data and used to relate the in vitro endpoint (t(50)) obtained in the study to a Draize MMAS (modified maximum average score). This provided a measure of the predicted in vivo eye irritation scores. In the current study, two separate laboratories used the same protocol to test the same set of coded materials and the results of both laboratories were compared to the initial PM. The TEA met the reliability criteria of the validation study in reproducing the predefined PM in both laboratories, and a good relationship between predicted and observed Draize MMAS values was obtained (r=0.906 and r=0.850). Good correlations were maintained when separate analyses were made of the formulations and ingredients included in the test set. Good relationships between the in vitro endpoint and individual Draize tissue scores (r>0.8) were also exhibited. Although insufficient data were available to make an assessment of interlaboratory variation, some difference in the reproducibility of the assay was noted between the two laboratories, particularly for the highly irritating materials. However, the consistency of data was encouraging and the discrepancies seen between the laboratories suggested a sensitivity of the model to subtle differences in application techniques, and in handling and timing. Taken together, these results indicate the utility of the TEA test for these types of substances and the need to more fully address the issue of interlaboratory reproducibility.

The Cytosensor(TM) microphysiometer assay and its associated prediction model were evaluated in the COLIPA ocular irritation validation study for cosmetic ingredients and formulations. Test materials were prepared in low-buffer medium and exposed to L929 cells grown in transwells. The metabolic rate of the cell population was measured after each dose and the dose inducing a 50% decrease in the rate (MRD(50)) was determined and used to predict the ocular irritation potential. Only 29 of the 55 materials could be tested because of solubility limitations. The irritancy potential of many chemical classes was underpredicted by the assay, particularly acids, bases and organics. The use of the assay for surfactants and surfactant-based formulations showed promise, confirming the use of the method for these types of materials, although some revision of the prediction model would be necessary. Excellent interlaboratory reproducibility for the MRD(50) values across all test materials was observed.

There is an urgent need to validate in vitro human skin models for use in safety testing. An important component of validation is characterizing the metabolizing capacity of these models. We report comparison of the expression of 139 genes encoding xenobiotic metabolizing enzymes in the EpiDerm model and human skin. In microarray analysis, the expression of 87% of the genes was consistent between the EpiDerm model and human skin indicating the presence of similar metabolic pathways suggesting commonality in function. Analysis of EpiDerm models constructed from four donors showed highly comparable expression of xenobiotic metabolizing genes demonstrating reproducibility of the model. Overall, the expression of Phase II enzymes appeared to be more pronounced in human skin and the EpiDerm model than that of Phase I enzymes, consistent with the role of skin in detoxification of xenobiotics. Though the basal expression of CYPs in particular was low in EpiDerm, significant induction of CYP1A1/1B1 activity was observed following treatment with 3-methylcholanthrene. These results indicate that the xenobiotic metabolizing capacity of the EpiDerm model appears to be representative of human skin. Models such as EpiDerm provide a valuable in vitro approach for evaluation of metabolism and toxicity of cutaneous exposures to xenobiotics.

Global gene expression profiling provides a useful means to identify key aspects of the skin aging process, and provides information to help develop new skin technologies. Important aspects of skin aging that can be addressed include skin hydration, barrier, matrix, pigmentation and antioxidant capacity. Human skin equivalent cultures allow topical application of test compounds, combinations and products to their stratum corneum surface and measurement of predictive biomarkers. Using this in vitro biomarker approach, it is possible to detect skin barrier enhancement in response to the compounds niacinamide and hexamidine, matrix effects to the peptides Pal-KT and Pal-KTTKS, and hydration and matrix responses to niacinamide and N-acetylglucosamine.

In spite of over 20 years of effort, no single in vitro assay has been developed and validated as a full regulatory replacement for the Draize Eye Irritation test. However, companies have been using in vitro methods to screen new formulations and in some cases as their primary assessment of eye irritation potential for many years. The present report shows the outcome of an Expert Meeting convened by the European Centre for the Validation of Alternative Methods in February 2005 to identify test strategies for eye irritation. In this workshop test developers/users were requested to nominate methods to be considered as a basis for the identification of such testing strategies. Assays were evaluated and categorized based on their proposed applicability domains (e.g., categories of irritation severity, modes of action, chemical class, physicochemical compatibility). The analyses were based on the data developed from current practice and published studies, the ability to predict depth of injury (within the applicable range of severity), modes of action that could be addressed and compatibility with different physiochemical forms. The difficulty in predicting the middle category of irritancy (e.g. R36, GHS Categories 2A and 2B) was recognized. The testing scheme proposes using a Bottom-Up (begin with using test methods that can accurately identify non-irritants) or Top-Down (begin with using test methods that can accurately identify severe irritants) progression of in vitro tests (based on expected irritancy). Irrespective of the starting point, the approach would identify non-irritants and severe irritants, leaving all others to the (mild/moderate) irritant GHS 2/R36 categories. © 2009 Elsevier Ltd.

The field of tissue engineering is multidisciplinary, seeking to fabricate artificial organs or substitutes in order to replace failing or damaged organs. These engineered tissues and organs are mostly targeted as substitutes for human donor tissues and as such are developed to avoid the complications associated with donor matching and immune rejection [1]. Examples include substitutes of organs including liver, heart, kidney, skin, teeth, and cornea [2].

Human skin equivalent cultures were investigated as possible pre-clinical skin irritation screens to aid safety assessments for chemicals and product formulations, and to facilitate design of safe and efficient human studies. In vitro responses in human skin equivalent cultures were compared directly to in vivo human skin responses from historic or concurrent skin tests for representative chemicals and products, including surfactants, cosmetics, antiperspirants, and deodorants. The in vivo data consisted of visual scores (i.e., erythema and edema) from skin-patch tests and diary accounts of skin irritation from product-use studies. In the in vitro studies, cornified, air-interfaced human skin cultures (EpiDerm) were evaluated using methods designed to parallel human clinical protocols with topical dosing of neat or diluted test substances to the stratum corneum surface of the skin cultures. The in vitro endpoints have previously been shown to be relevant to human skin irritation in vivo, including the MTT metabolism assay of cell viability, enzyme release (lactate dehydrogenase and aspartate aminotransferase), and inflammatory cytokine expression (Interleukin-1alpha). For surfactants, dose-response curves of MTT cell-viability data clearly distinguished strongly-irritating from milder surfactants and rank-ordered irritancy potential in a manner similar to repeat-application (3x), patch-test results. For the antiperspirant and deodorant products, all the in vitro endpoints correlated well with consumer-reported irritation (r, 0.75-0.94), with Interleukin-1alpha (IL-1alpha) release, showing the greatest capacity to distinguish irritancy over a broad range. IL-1alpha release also showed the best prediction of human skin scores from 14-day cumulative irritancy tests of cosmetic products. These results confirm the potential value of cornified human skin cultures as in vitro pre-clinical screens for prediction of human skin irritation responses. A preliminary report of these results has been published.

A human cell-based in vitro method was developed to screen for ocular irritancy potential of aqueous compatible and incompatible test agents, such as liquids, insoluble solids, powders, granulars, emulsions, and acids/alkalis. Methods were developed for topical application (an exposure that mimics in vivo testing) and wash-off of test substances on the epithelial surface of human skin derived epithelial-fibroblast cocultures (Skin2 Model ZK1200 from Advanced Tissue Sciences). These cultures contain noncornified stratified squamous epithelium, providing a three-dimensional in vitro model that resembles noncornified mucosal epithelium, such as cornea and conjunctiva. The hypothesis tested and confirmed in this work was that the rate of cytotoxicity induced by topical application of test substances to the stratified epithelial cell cultures would correlate with ocular irritancy. Test substances were applied to the cell system for up to 30 min, and cytotoxicity was measured as decreased 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) vital dye metabolism. The time (in minutes) of exposure to test agent that reduced MTT metabolism to 50% of control levels (the t50 value) was calculated for each test substance, and these values showed a good correlation (r = 0.87) with historic rabbit low-volume eye test maximum average scores (MAS) for a range of liquid, solid, granular, powder, and other test materials, including consumer product formulations and ingredient chemicals. Additionally, t50 values confirmed the mild nature of selected cosmetic formulations. Taken together, these data indicate that this in vitro test, termed the tissue equivalent assay, is a valuable tool to screen for irritancy of test substances to mucosal tissues, such as cornea and conjunctiva.

It has been proposed [J. D. McKinney, J. Fawkes, S. Jordan, K. Chae, S. Oatley, R. E. Coleman, and W. Briner (1985). Environ. Health Perspect. 61, 41-53] that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces toxic responses through persistent occupancy of nuclear thyroxine (T4) receptors, and that maintenance of receptor occupancy by supraphysiologic concentrations of thyroid hormones mimics TCDD toxicity [L. H. Hong, J. D. McKinney, and M. I. Luster (1987). Biochem. Pharmacol., 36, 1361-1365]. TCDD induces hyperkeratinization in cultured normal human epidermal cells and the human keratinocyte line, SCC-12F. This response is associated with a decrease in high-affinity epidermal growth factor (EGF) receptors. These cell systems were used as models to compare the actions of TCDD with those of triiodothyronine (T3) and T4 on human target cells. Keratinocytes were treated simultaneously with T3 and T4 in a 4:1 molar ratio (T3/T4; Hong et al., 1987) and levels of EGF binding and 7-ethoxycoumarin O-deethylase activity (a marker for cytochrome P1-450 induction) were measured. T3/T4 (at concentrations up to 10 microM T3/2.5 microM T4) and T3 or T4 alone (0.1 to 10 microM) did not produce the hyperkeratinization, the decrease in EGF binding, or the increase in ECOD activity that are characteristic of TCDD exposure. Nonresponsiveness to T3/T4 was not due to metabolism of these hormones by the keratinocytes. T3 and T4 did not compete with [3H]TCDD for binding to cytosolic Ah receptor from C57BL6 mouse liver, SCC-12F, or normal human epidermal cells. TCDD and an active stereoisomer, 2,3,7,8-tetrachlorodibenzofuran, did not compete with [125I]T3 or [125I]T4 for binding to nuclear receptors from SCC-12F cells or C57BL6 mouse liver. Taken together, these data demonstrate that the actions of TCDD and thyroid hormones are mediated by distinct mechanisms in human keratinocytes.

Hydrocortisone (cortisol) increased the binding of thyrotropin-releasing hormone (TRH) to specific membrane receptors in 4 clonal strains of rat pituitary cells. At the highest effective cortisol concentration (3–5 × 10 −6 M), the increase was observed within 6–8 hr and became maximal (140 to 160% of control binding) by 18–24 hr. Half-maximum stimulation occurred in serum-containing medium at 9 × 10 −8 M cortisol, and a significant increase in TRH binding was seen at 3 × 10 −8 M. Equilibrium binding studies showed that enhanced TRH binding was explained by an increase in receptor number with no change in affinity. Similar effects were seen with Dexamethasone, but no increase in TRH binding was noted when testosterone, methyltestosterone, progesterone, estradiol or the antiestrogen Lilly 88571 were added to the culture medium. Cortisol treatment did not cause the appearance of specific TRH binding sites in cell strains previously shown to lack receptors for the tripeptide (F 4 C 1 , GH 1 2C 1 and R 5 cells). When added cortisol was removed from medium, receptor number decayed to control values with a T 1 2 of about 30 hr. Previous studies have shown that TRH receptors in GH-cells can be down-modulated by TRH and thyroid hormones; the present findings demonstrate that glucocorticoid hormones can increase the number of TRH receptors in GH-cells.

GH4C1 cells are strain of rat pituitary cells which synthesize and secrete both PRL and GH. Phorbol esters, which are potent tumor promoters in mouse skin and have diverse actions in a variety of cell and tissue systems, were found to alter hormone production by GH4C1 cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA), the most potent phorbol ester, stimulated both PRL synthesis and release. The synthesis of PRL was stimulated 2.3- to 6.2-fold over control values after 3 days of treatment; the ED50 for TPA was 8.9 +/- 2.1 nM (5.5 +/- 1.3 ng/ml). The release of stored PRL was stimulated within 10 min of the addition of 100 ng/ml TPA to the culture medium. Maximum stimulation of release was 1.3- to 2.3-fold over control, and the ED50 for TPA was 13.2 +/- 2.3 nM (8.1 +/- 1.4 ng/ml). Another tumor-promoting phorbol ester, phorbol 12,13-didecanoate (PDD), stimulated PRL synthesis and release to the same maximum extent at TPA, but two biologically inactive compounds, 4 alpha-PDD and phorbol, had no effect. The stimulation of PRL release by TPA was a Ca++-dependent process; Co++, a competitive antagonist of Ca++, at 2.0 mM blocked completely the stimulation of PRL release by 100 ng/ml TPA, and this block was overcome by 2.0 mM Ca++, but not by 2.0 mM Mg++. Although phorbol esters stimulate prostaglandin (PG) synthesis in certain other cells types, PGs were not required for TPA action in GH4C1 cells. Indomethacin, a PG cyclooxygenase inhibitor, at 200 ng/ml did not inhibit the stimulation of PRL synthesis and release by TPA. Furthermore, there was no detectable (less than 50 pg/ml) PGE2 or PGF2 alpha in the medium of control or TPA-treated cells. In GH3 cells, a related cell strain, TPA (100 ng/ml) decreased GH production in both control and hydrocortisone-stimulated cells to 35% of untreated control values after 3 days of incubation. TPA also altered cell morphology, but had little or no effect on cell growth. We conclude that GH cells provide a useful system to study the mechanisms of phorbol ester action on differentiated functions.

Human exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and chlorinated analogs commonly results in pathological changes in the skin and its appendages characterized by thickening of the epidermis (acanthosis), hyperkeratosis and squamous metaplasia of the epithelial lining of the sebaceous glands. Acneform lesions (chloracne) develop as hair follicles dilate and fill with keratin and sebaceous glands become cystic. In animal models it has been found that the chloracneogenic potential of the halogenated aromatic compounds examined corresponds with the relative affinity of these same compounds for the cytosolic TCDD receptor. This receptor controls the coordinate expression of a number of inducible enzyme activities and in certain cell targets can alter normal programs of proliferation and differentiation. In this report we describe some of our ongoing studies on the mechanisms of action of TCDD in normal human epidermal cells and squamous cell carcinoma (SCC) lines. These systems permit detailed investigation of the molecular and biochemical events underlying pathologic changes in the skin and offer the potential of establishing a risk assessment model for halogenated aromatic compounds by using human target cells. Images FIGURE 2. A FIGURE 2. B FIGURE 2. C FIGURE 2. D FIGURE 3.

2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD) acts on selected targets within the immune system to produce a characteristic profile of pathologic responses typified by thymic atrophy, suppressed cellular immunity, and inhibition of antibody production to T-lymphocyte-dependent antigens. Studies in inbred mice differing in sensitivity to TCDD indicate that TCDD-induced thymic atrophy is mediated by a receptor protein (designated the Ah receptor). To study the cellular and molecular basis for TCDD-induced thymic atrophy, primary cultures of thymic epithelial (TE) cells were established from C57BL 6 mice, a strain sensitive to TCDD. Treatment of TE monolayers with TCDD (0.1 to 10 n m ) resulted in the altered maturation of cocultured syngeneic thymocytes as judged by suppression (40% of control at 10 n m TCDD) of TE-dependent responsiveness of thymocytes to the mitogens concanavalin A and phytohemagglutinin. TE-conditioned medium enhanced the mitogen responsiveness of thymocytes three- to four-fold; however, the enhanced mitogen response mediated by the TE-conditioned medium was not suppressed in thymocytes incubated in medium collected from TCDD-treated cultures or in TE-conditioned medium to which TCDD (10 n m ) had been added directly. The suppression of TE-dependent maturation of thymocytes was concentration dependent (EC 50 ∼ 1 n m ) and stereospecific, suggesting involvement of the Ah receptor. The Ah receptor in cytosol fractions from cultured TE cells was measured directly and was found to be present at a concentration 3 and 3.5 times greater than that measured in whole thymus and thymocytes, respectively. The results of this study indicate that TCDD can act directly on epithelial target cells in the thymus: one consequence of this action appears to be the altered thymus-dependent maturation of T-lymphocyte precursors, mediated through direct cell-cell contact between thymocytes and TE cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and isosteric halogenated analogs produce a spectrum of pathologic changes in the epidermis of humans. In this study, the actions of TCDD on cultured human epidermal cells were characterized to determine whether these cells are an appropriate in vitro model to examine the mechanisms of TCDD toxicity to human skin. The differential staining properties of TCDD-treated cultures indicated that TCDD decreased basal cell numbers and increased the degree of keratinization. Histologic examination of cross-sections of the cultures confirmed a loss of small nucleated cells and increased cell layering in response to TCDD. TCDD produced no change in total cell number or cell protein, but decreased the number of small (basal) cells and DNA synthesis. TCDD increased the number of cells containing spontaneous envelopes, as well as the number of envelope-competent cells. The quantitative changes observed in these parameters were consistent with a TCDD-induced commitment of proliferating cells to terminal differentiation. TCDD also decreased epidermal growth factor (EGF) specific binding. Maximal changes in EGF binding occurred after 4 days, and in small cell number after 5 days. The decreases in EGF binding and small cell number were stereospecific and concentration dependent (EC50, 1 to 2 nM), implicating the human Ah receptor in mediating these responses to TCDD. These data indicate that TCDD treatment produces hyperkeratinization in cultured human epidermal cells. It is proposed that TCDD acts on epidermal basal cells to enhance terminal differentiation through mechanisms regulated at least in part by the Ah receptor.

It was recently reported that suppression of murine bone marrow hematopoiesis is a very sensitive indicator for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity (1). We report here that a structural analog of TCDD, 1-NH2-3,7,8-trichlorodibenzo-p-dioxin (NH2-TriCDD), is a specific and effective antagonist for TCDD-induced myelotoxicity and enzyme induction. When administered to mice or added directly into culture at a 100-fold excess, relative to TCDD, NH2-TriCDD completely abrogated the ability of TCDD to inhibit granulocyte-macrophage progenitor cells (CFU-C) formation, an indicator of hematopoiesis. Further, NH2-TriCDD inhibited TCDD-induced activation of cytochrome P1-450 monooxygenase activity. Studies designed to measure specific binding of TCDD to the cytosolic Ah receptor indicated that NH2-TriCDD effectively inhibited binding of TCDD to the receptor by acting as a competitive antagonist (Ki = 0.72 nM).