keratinocytes

/Tag:keratinocytes

Interleukin-1α and Interleukin-8 Release by Human Keratinocyte Cell Culture Treated with Surfactants

Michio Shibata, Takanari Tsuda, Hiroshi Itagaki, Shinobu Kato,
Toshiaki Kobayashi, Hideyuki Ichikawa and Yoshihiro Morikawa

The effects of four cosmetic surfactants on interleukin (IL)-1α and IL-8 release from human keratinocytes were studied to investigate the feasibility of using these effects for the prediction of the irritation potential of chemicals. After exposure of cells to surfactants, the amounts of IL-1α and IL-8 released into culture medium were measured by ELISA. Cytotoxicity was evaluated by using the neutral red uptake (NRU) cytotoxicity assay. Cytokine release was increased 7–15 times by sodium lauryl sulphate (SLS), laurtrimonium chloride, cocamidopropyl betaine (CPB) and Oleth-5 at cytotoxic concentrations. IL-8 release was increased 3–4 times by SLS, CPB and Oleth-5 at subcytotoxic concentrations. After exposure to SLS, IL-1α was released within 1 hour, suggesting that IL-1α release is associated with membrane damage, whereas IL-8 release continued for 24 hours, suggesting that IL-8 was produced within the cells. Cytotoxicity tests and IL-8 release assays were also performed on seven other surfactants. The results show that moderate irritants CPB and PEG-4 dioleate, which have weak cytotoxic effects, significantly increased IL-8 release from human keratinocytes. It is suggested that measurement of IL-8 release is useful for predicting the irritation potential of chemicals which cannot be detected by using the NRU cytotoxicity assay.
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Alternative Methods for Skin Irritation Testing: the Current Status

Philip A. Botham, Lesley K. Earl, Julia H. Fentem, Roland Roguet and Johannes J.M. van de Sandt

The ECVAM Skin Irritation Task Force was established in November 1996, primarily to prepare a report on the current status of the development and validation of alternative tests for skin irritation and corrosion and, in particular, to identify any appropriate non-animal tests for predicting human skin irritation which were sufficiently well-developed to warrant ECVAM supporting their prevalidation/validation. The task force based its discussions around the proposed testing strategy for skin irritation/corrosion emanating from an OECD workshop held in January 1996. The following have been reviewed: a) structure-activity and structure-property relationships for skin corrosion and irritation; b) the use of pH and acid/alkaline reserve measurements in predicting skin corrosivity; c) in vitro tests for skin corrosion; d) in vitro tests for skin irritation (keratinocyte cultures, organ cultures, and reconstituted human skin models); and e) human patch tests for skin irritation. It was apparent that, although several promising candidate in vitro tests for skin irritation (for example, reconstituted human skin methods, and human and animal skin organ culture methods) were under development and evaluation, a test protocol, a preliminary prediction model and supporting data on different types of chemicals were only available for a method employing EpiDermTM. Thus, it is proposed that this EpiDerm test undergoes prevalidation during 1998. In addition, since it was felt preferable to be able to include other in vitro tests in such a prevalidation study, it is recommended that a “challenge” be set to anyone interested in taking part. This involves submitting data on ten test chemicals selected by the task force, obtained according to a standard protocol with a preliminary prediction model, for review by the task force by 31 May 1998.
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Selective Induction of Interleukin-12 in Reconstructed Human Epidermis by Chemical Allergens

Emanuela Corsini, Elena Limiroli, Marina Marinovich, Catherine
Cohen, Roland Roguet and Corrado L. Galli

Keratinocytes play an important role in skin inflammatory and immunological reactions through the release of cytokines and response to them. These cells have been shown to direct T-cell priming by producing cytokines such as interleukin (IL)-10 and IL-12. The purpose of this work was to explore the potential use of IL-12 production to discriminate between skin irritants and contact allergens in vitro. Initially, a reconstituted human epidermis was treated with a known human skin irritant, sodium lauryl sulphate (SLS), and a known human contact allergen, 1-chloro-2,4-dinitrobenzene (DNCB). The expression of IL-12p40 was assessed at specific time intervals by the semi-quantitative reverse transcriptase-polymerase chain reaction (rt-PCR). The data obtained indicated that only DNCB induced an up-regulation of IL-12p40. This up-regulation occurred after exposure to DNCB for 3 hours. Importantly, the application of SLS or vehicles did not induce IL-12 mRNA up-regulation. An increase in total IL-12 protein content was detected in supernatants of allergen-stimulated, but not vehicle-stimulated, reconstituted epidermis. To confirm these results, the effects of benzalkonium chloride, oxazolone and eugenol were assessed. At concentrations that resulted in equivalent IL-1α release, only contact allergens increased IL-12 expression, which confirmed the previous results. These data suggest that IL-12, which is crucial for T-helper type 1 cell responses, could be a useful marker for discriminating between contact allergens and irritants.
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Iron-induced Lipid Peroxidation in Human Skin-derived Cell Lines: Protection by a Red Orange Extract

Flora Morini, Fabiola Dusatti, Francesco P. Bonina, Antonella Saija and Margherita Ferro

Although photodamage and photoprotection have already been extensively studied in cultured cells, few data have been reported in the literature regarding the in vitro behaviour of skin cells toward a chemical stress, such as iron-induced lipid peroxidation. We investigated the susceptibilities of two human skin-derived cell lines (NCTC 2544 keratinocytes and HFFF2 fibroblasts) to lipid peroxidation induced by FeSO4/histidine, FeSO4/ascorbate and Fe2(SO4)3/ADP. NCTC 2544 cells were more susceptible than HFFF2 cells to lipid peroxidation (assessed by measuring the content of malondialdehyde [MDA]) with iron/ascorbate and iron/ADP as pro-oxidants whereas, with iron/histidine, the same level of MDA production was achieved (about 10nmol/mg protein) in the two cell populations. On the basis of these results, one experimental model (iron/histidine) was selected to assess the protective effect of a mixture of two classical antioxidants, Trolox C™ (50μM) and Vitamin C (1mM), added to the cell cultures according to various protocols. The maximal decrease of MDA production in both cell lines was obtained when the antioxidant mixture and the pro-oxidant were added simultaneously to the cultures. By using the same experimental design, NCTC 2544 and HFFF2 cells were exposed to a standardised extract of red oranges (ROE; 0.025–0.5mg/ml), the main active principles of which (anthocyanins 3.1%, hydroxycinnamic acid 2.07%, and flavanone glycosides 8.1%) possess antioxidant activity. Cells treated with ROE, that were still over 90% viable, as evaluated by means of neutral red uptake and tetrazolium salt reduction tests, showed a significant and dose-dependent inhibition of MDA production. This study provides new information about the behaviour of cultured skin cells exposed to chemically induced oxidative stress, and provides further support to the possibility of using skin-derived human cell lines in the evaluation of the effectiveness of antioxidant ingredients for new drugs and/or cosmetics.
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The Development of a Standardised Protocol to Measure Squamous Differentiation in Stratified Epithelia, by using the Fluorescein Cadaverine Incorporation Technique

Alison C. Gray, Joanne Malton and Richard H. Clothier

Fluorescein cadaverine (FC) incorporation into cornified envelopes during squamous differentiation in stratified epithelia acts as a fluorescent substitute for endogenous transglutaminase substrates that can be visualised and quantified. The FC incorporation technique has been used to evaluate squamous differentiation in keratinocytes cultured in a medium that stimulates differentiation and in response to modulation by chemicals. A Standard Operating Procedure for the measurement of squamous differentiation is required as part of the prevalidation procedure for in vitro assays. In the present study, keratinocytes were isolated from the epidermis of 34 human donors. Cellular metabolic activity (resorufin production), total protein (kenacid blue uptake) and transglutaminase activity (FC incorporation) were measured in 87 and 21 independent runs at 6 and 12 days, respectively. Analysis of the control data showed that the cultures had a mean resorufin production that decreased over 12 days. This was inversely related to FC incorporation, which increased over 12 days. Mean protein concentration was reduced over the 12 days, but not in analyses that used donors for whom both 6-day and 12-day data were available. This information was used to define the normal limits within which the data should fall (mean ± 1 SD). Data sets falling outside the normal limits performed statistically no differently from the normal responders, in experiments conducted to investigate the effects of chemicals on the modulation of squamous differentiation in keratinocytes. This was demonstrated by using compounds that modify transglutaminase expression and/or activity. All-trans retinoic acid significantly inhibited FC incorporation, but stimulated resorufin production at 1 × 10–7M and above. Nicotine significantly up-regulated both FC incorporation and resorufin production at 125μg/ml. Hence, it was concluded that this robust assay approach, in which keratinocytes from a range of donors respond predictably to the test chemicals employed, did not justify the limitations that would be imposed by setting criteria that eliminated all data lying outside the normal range.
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Evaluation of the Cytotoxic Effects of Humid Lightweight Coal Ash derived from the Disposal of Waste on Normal Human Keratinocyte and Endothelial Cell Lines in 2-D and 3-D Culture

Chiara Scanarotti, Stefania Vernazza, Massimiliano Brignone, Jenia Danailova, Maria A.
Pronzato and Anna M. Bassi

The presence of waste in the environment has frequently been indicated as a significant risk to human health. Therefore, landfill sites and the disposal of urban solid and non-hazardous waste by incineration are subject to much environmental monitoring, in addition to the regulations already in place.
However, little action has been taken, and consequently no specific legislation exists, in relation to the assessment of the real biological risk of various substances, including chemical mixtures and ashes, derived from the incineration processes. This study assessed the cytotoxic potential of humid lightweight coal ash (LA) derived from incineration processes and waste management, on two cell lines: NCTC 2544 normal human keratinocytes and HECV endothelial cells. To reach this goal and to assess more-realistic methods for animal replacement, we employed different in vitro experimental approaches: acute and longer exposure to LA, by direct and indirect contact (0–2mg/ml and 16mg, respectively), both in 2-D and 3-D cultures.
In 2-D HECV cultures, we observed a decrease in the viability index, but only during direct contact with LA doses higher than 0.1mg/ml. Moreover, some striking differences in cytotoxicity were observed between the 2-D and 3-D models. Taken together, these observations indicate that, for studying pollutant toxicity during longer exposure times, 3-D cultures in direct contact with the pollutant seem to offer a more suitable approach — they mimic the in vivo behaviour of cells more realistically and under strictly controlled conditions. Thus, in readiness for possible forthcoming European regulations, we believe that the proposed study, even in its preliminary phase, can provide new advice on the assessment of the toxic and biological potential of particular chemical compounds derived from waste management processes.
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