This Article Abstract Free Full Text (Free PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Varani, J. Articles by Dunstan, R. Search for Related Content PubMed PubMed Citation Articles by Varani, J. Articles by Dunstan, R. Social Bookmarking                   What's this?

Human Skin in Organ Culture and Human Skin Cells (Keratinocytes and Fibroblasts) in Monolayer Culture for Assessment of Chemically Induced Skin Damage

¹ Department of Pathology, The University of Michigan Medical School, Ann Arbor, Michigan 48109
² Pfizer Global Research and Development, Ann Arbor, Michigan 48105

Correspondence: Address correspondence to: James Varani, Ph.D., Department of Pathology, The University of Michigan, 1301 Catherine Road/Box 0602, Ann Arbor, MI 48109, USA; e-mail:varani{at}umich.edu

	Abstract

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Human skin cells (epidermal keratinocytes and dermal fibroblasts) in monolayer culture and human skin in organ culture were exposed to agents that are known to produce irritation (redness, dryness, edema and scaly crusts) when applied topically to skin. Among the agents used were three well accepted contact irritants (i.e., all-trans retinoic acid [RA], sodium lauryl sulfate [SLS] and benzalkonium chloride) as well as the corrosive organic mercury compound, aminophenyl mercuric acetate (APMA), and 5 contact sensitizers (oxazolone, nickel sulfate, eugenol, isoeugenol and ethylene glycol dimethacrylate [EGDM]). As a group, the contact irritants (including the corrosive mercuric compound) were cytotoxic for keratinocytes and fibroblasts and suppressed growth at lower concentrations than the contact sensitizers. The contact irritants also produced histological changes (hyperplasia, incomplete keratinization, loss of the granular layer, acantholysis and necrosis) in organ-cultured skin at dose levels at which the contact sensitizers appeared to be inert. Finally, the profile of secreted molecules from organ-cultured skin was different in the presence of contact irritants versus contact sensitizers. Taken together, these data suggest that the use of organ-cultured skin in conjunction with cells derived from the skin in monolayer culture may provide an initial approach to screening agents for deleterious changes in skin.

Key Words: Skin • organ culture • keratinocyte • fibroblast • irritation • contact irritant • contact sensitizer

Abbreviations: RA, All-trans retinoic acid • SLS, Sodium lauryl sulfate • APMA, Aminophenyl mercuric acetate • EGDM, Ethylene glycol dimethacrylate • KBM, Keratinocyte basal medium • KGM, Keratinocyte growth medium • DMEM, Dulbecco’s modified minimal essential medium • FBS, Fetal bovine serum • EGF, Epidermal growth factor • IL, Interleukin • ELISA, Enzyme-linked immunosorbant assay

	Introduction

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Agents developed for formulation as cosmetics/cosmeceuticals need to be evaluated for detrimental effects on the skin. Topical pharmaceuticals also need to be evaluated for the same effects as does the large group of agents in the "household products" category. The goal of safety-assessment is to define the theoretical benefit of a compound compared to what damage, if any, the compound may cause. Safety assessment is currently based on clinical evidence of skin damage using animal models developed decades ago (Draize et al., 1944). However, several alternatives to animal testing have recently been or are currently being evaluated. In part, these efforts to eliminate or reduce animal use in testing are based on ethical considerations and/or marketing strategies and cost. In addition, however, it is recognized that results obtained in animal tests are not always predictive of the response of human skin (Nixon et al., 1975; York et al., 1996).

Among the alternatives being evaluated are three-dimensional organotypic cultures consisting of epidermal keratinocytes on a collagenous matrix (with or without imbedded dermal fibroblasts) and whole skin organ cultures from rodents, pigs or, in some cases, discarded human skin (Botham et al., 1998; Boelsma et al., 2000; Ponec et al., 2002; Jacobs et al., 2004; Welss et al., 2004). Multiple endpoints have been examined with the various culture systems in an effort to find functional and/or biochemical changes that are predictive of skin damage. To date, however, validated assays are only available to confirm corrosivity. No assay has proven capable of achieving all of the requirements needed to replace testing in animals (Botham, 2004). In the present report, we describe results using a combination of skin cells (human epidermal keratinocytes and human dermal fibroblasts) in monolayer culture and adult human skin in organ culture to identify and characterize changes in structure/function following exposure to known corrosive, irritant and sensitizing agents.

	Materials and Methods

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Chemical Agents
The organic mercury compound, aminophenyl mercuric acetate (APMA), was used as a corrosive agent. Irritants included the detergent, sodium lauryl sulfate (SLS), the biologically-active retinoid, all-trans retinoic acid (RA) and benzalkonium chloride. Five agents that are known to be human skin contact-sensitizers were also examined. These included oxazolone (classified as a strong sensitizer), nickel sulfate (strong), isoeugenol (strong), eugenol (moderate) and ethylene glycol dimethacrylate (EGDM; moderate) (Basketter et al., 2005).

Organ Culture
Replicate 2 mm punch biopsies were obtained from hip skin of volunteers less than 70 years of age. The participation of human subjects in this study was approved by the University of Michigan Institutional Review Board and all subjects provided written informed consent prior to their inclusion in the study. The punch biopsies were incubated in wells of a 24 well dish (1 tissue piece per 250 µl of culture medium). Culture medium consisted of Keratinocyte Basal Medium (KBM) (Cambrex Biologicals, Walkersville, MD). KBM is a modification of MCDB-153 medium. For our purposes, it was supplemented with calcium chloride to a final Ca²⁺ concentration of 1.4 mM. One or 2 tissue pieces were left as controls while the others were treated with the irritants or allergens over a range of concentrations. Fresh culture medium was provided at 2-day intervals.

Organ culture-conditioned medium collected on day 2 was saved for assessment of growth factors, cytokines and type I procollagen levels as described below. At the end of the incubation period (day 8), organ-cultured tissue was fixed in 10% buffered formalin and embedded in paraffin. Five-µm-thick sections were cut and stained with hematoxylin and eosin. Representative sections of each biopsy were selected for histological evaluation and examined. In certain experiments, tissue was fixed at day 2 and stained with methyl green pyronine (Jacobs et al., 2002). Human skin in organ culture has been extensively used in the past. The protocol used here was virtually identical to that described in our past reports (Varani et al., 1993, 1994).

Cells in Monolayer Culture
Human epidermal keratinocytes and human dermal fibroblasts were used in these studies. Keratinocytes were isolated from neonatal foreskin tissue obtained at circumcision (Varani et al., 1994). They were maintained in Keratinocyte Growth Medium (KGM). KGM consistes of the same basal medium as KBM but is supplemented with epidermal growth factor (EGF), insulin and pituitary extract. Cell growth was at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air. Keratinocytes were subcultured using trypsin/EDTA as required, and used at passage 3 to 5. An immortalized keratinocyte cell line (Hacat) (Boukamp et al., 1988) was also utilized as the epithelial cell component in some of the studies. These cells were handled exactly as low-passage keratinocytes and used interchangeably with those cells.

Fibroblasts were isolated from the same tissue as used for the isolation of keratinocytes (Varani et al., 1994). Fibroblasts were maintained in Dulbecco’s MEM supplemented with non-essential amino acids and 10% fetal bovine serum (DMEM-FBS). Cell growth was at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air. Fibroblasts were subcultured using trypsin/EDTA as required, and used at passage 3 to 5.

Cytotoxicity and Growth Inhibition Assays
Keratinocytes and fibroblasts were added to wells of a 24-well dish at 8 x 10⁴ cells per well using their respective growth media (KGM for keratinocytes and DMEM-FBS for fibroblasts). One day later, after the cells had attached and spread, the culture medium was removed and the cells washed 1 time in serum-free medium. At this point, 0.5 ml of KGM was added to keratinocyte cultures. The same medium was used for fibroblasts with the exception that it was supplemented with extracellular Ca²⁺ to bring the final concentration to 1.4 mM (Varani et al., 1994). The agents to be examined were added to the wells of both cell types over a wide range of concentrations. Cells were incubated for 4 hours and then harvested with trypsin/EDTA and counted. Following this, the cells were replated in wells of a 24-well dish using DMEM-FBS for both cell types. The number of cells that reattached and spread was determined four hours later. Cells capable of successfully "replating" were assumed to be viable and functional (Varani et al., 1992).

To assess growth inhibition, keratinocytes and fibroblasts were added to wells of a 24-well dish and treated exactly as above. Cells were incubated for 48 hours and then harvested with trypsin/EDTA and enumerated.

Assessment of Secreted Molecules
Organ culture fluids collected at day 2 were analyzed for levels of amphiregulin by enzyme-linked immunosorbant assay (ELISA) using a commercially available kit (R&D Systems; Minneapolis, MN) as described previously (Rittie et al., 2006). The same organ culture fluids were assayed for type I procollagen levels using a commercially available ELISA kit (Pan Vera Corp., Madison, WS) (Varani et al., 2004). Levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) were quantified as part of a multiplex assay (Luminex antibody bead kit; BioSource International, Inc.; Camarillo, CA). Results from the multiplex assay were confirmed using individual ELISA kits for the 2 cytokines (Upstate Biologicals; Charlottesville, VA and Biosource International, Inc.).

Statistical Analysis
Differences between groups in experiments with multiple groups were analyzed for statistical significance by ANOVA followed by paired-group comparisons. Differences from control values at the p < 0.05 level were considered significant. Correlation coefficients were determined in experiments designed to assess relationships between levels of different metabolites in a given sample.

	Results

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Keratinocyte and Fibroblast Cytotoxicity and Growth Inhibition: Comparison of Chemicals with Corrosive, Irritant and Allergenic Potential
In the first series of experiments, a panel of 3 contact irritants and 5 contact sensitizers was examined for cytotoxicity with human epidermal keratinocytes and human dermal fibroblasts in monolayer culture. A corrosive organic mercury compound (APMA) was examined in the same assay. The 3 irritants demonstrated minimal (or no) cytotoxic activity at concentrations up to 1 µM while the organic mercury compound was cytotoxic to virtually 100% of the keratinocytes and fibroblasts at 1 µM and killing of both cell types (30–40% killed) was seen at concentrations as low as 0.1 µM (Figure 1). In contrast, there was no toxicity with any of the 5 contact sensitizers at concentration below 100 µM (Figure 1).

View larger version (15K): [in this window] [in a new window]   Figure 1 Keratinocyte and fibroblast killing by contact irritants and contact sensitizers in the 4-hour cytotoxicity assay. Values are means (percent killed) ± standard errors based on duplicate samples in 3 separate experiments with each cell type. Statistical significance was determined by ANOVA followed by paired-group comparisons. With both cell types, APMA induced statistically significant cytotoxicity at 0.1 µM. RA and benzalkonium chloride were statistically different from control at 5 µM. EGDM was statistically different from control keratinocytes at 100 µM and fibroblasts at 500 µM. Eugenol was statistically different with keratinocytes at 250 µM and fibroblasts at 500 µM.

View larger version (16K): [in this window] [in a new window]   Figure 2 Effects of contact irritants and contact sensitizers on keratinocyte and fibroblast proliferation in a 2-day growth assay. Proliferation index is the number of cells at day 2 divided by the number at time-zero. Values are means ± standard errors based on duplicate or triplicate samples in a total of 5–7 experiments with each cell type. Statistical significance was determined by ANOVA followed by paired-group comparisons. With keratinocytes, RA and benzalkonium chloride were statistically different from control at 1 µM, SLS at 5 µM and nickel sulfate and EGDM at 500 µM. With fibroblasts, RA and benzalkonium chloride were statistically different from control at 5 µM, SLS at 10 µM, EGDM at 100 µM and nickel sulfate at 500 µM.

View larger version (68K): [in this window] [in a new window]   Figure 3 Histological features of human skin after incubation for 8 days in organ culture in the presence of different contact irritants. Epidermal hyperplasia can be seen in tissues treated with RA and, to a lesser extent, with SLS but not with benzalkonium chloride. With increasing concentrations of RA there are abnormalities in the upper epidermis including incomplete keratinization, loss of the granular layer, acantholysis and separation of the upper layers from the lower layers of epidermal cells. With SLS, necrosis of the upper epidermis is seen under conditions in which the lower layers of cells remain intact and viable. In contrast, at 10 µM benzalkonium chloride, complete necrosis throughout the epidermis is seen. All sections are hematoxylin and eosin stained (original magnification x133). Each agent was examined with tissue from 9–14 different individuals with similar resutls.

Figure 3 also demonstrates histological changes in organ-cultured skin treated with benzalkonium chloride. There were no consistent changes in tissue exposed to 1 µM benzalkonium chloride. At 5–10 µM, epidermal necrosis was observed. With benzalkonium chloride treatment, necrosis was observed throughout the entire tissue, including the basal layer. This was in contrast to what was seen with RA and SLS, where the major effects were on the upper (differentiating) layers of the epidermis. Finally, organ-cultured skin was exposed to 1 µM APMA for 8 days. Complete necrosis throughout the epidermis and dermis was observed in every specimen (not shown). Histological changes induced by APMA and benzalkonium chloride were indistinguishable, except for the rapidity with which they developed and the dosage. Skin organ cultures were also treated with increasing concentrations of the five contact sensitizers. Concentrations as high as 100–250 µM did not cause necrosis with any of the agents. In some tissues, epidermal thinning was observed at the highest concentration (500 µM). However, even at 500 µM, most of the tissue samples were indistinguishable from controls (not shown).

In an effort to detect cytotoxic changes in the tissue before overt necrosis was apparent, we stained sections of irritant-treated, organ-cultured skin with methyl green-pyronine after incubation for 2 days. With irritant concentrations that eventually produced overt cytotoxicity (i.e., 10 µM RA, SLS and benzalkonium chloride), there was a loss of RNA (decreased methyl green-pyronine staining) evident at day 2. However, decreased staining (relative to freshly biopsied skin) was also seen in sections of control tissue. The loss of RNA in the control tissue was never as great as seen following exposure to the irritants, but differences between treated and control skin were subtle (data not shown).

Amphiregulin and Type I Procollagen Production in Organ-Cultured Skin Exposed to Contact Irritants and Contact Sensitizers
Organ culture fluids obtained 2 days after treatment with each of the 3 irritants and 5 sensitizers were analyzed for amphiregulin. The amphiregulin level was significantly higher in culture fluid from RA-treated skin than control skin (Figure 4). Amphiregulin in organ culture fluid from SLS-treated tissue was also elevated, but the increase with SLS, although statistically significant, was not as dramatic as seen with RA. In contrast, neither benzalkonium chloride nor any of the 5 sensitizers had a significant effect on amphiregulin production (Figure 4). Thus, the increase in amphiregulin correlated with epidermal hyperplasia rather than with necrosis, per se.

View larger version (15K): [in this window] [in a new window]   Figure 4 Amphiregulin levels in skin organ cultures. The three contact irritants were examined at the concentrations shown while the values presented for the contact sensitizers were from biopsies treated with each agent at a concentration of 250 µM. Values shown represent means ± standard errors based on organ cultures from 6–14 individuals per test agent. Control values have been normalized to 1.0 and the other values expressed in relation to this. * indicates statistical differences from the control value (p < 0.05).

View larger version (15K): [in this window] [in a new window]   Figure 5 Type I procollagen in skin organ cultures. The 3 contact irritants were examined at the concentrations shown while the values presented for the contact sensitizers were from biopsies treated with each agent at a concentration of 250 µM. Values shown represent means ± standard errors based on organ cultures from 6–14 individuals per test agent. Control values have been normalized to 1.0 and the other values expressed in relation to this. * indicates statistical differences from the control value (p < 0.05).

	Discussion

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Redness, dryness, edema, and scaly crusts are common manifestations of chemically induced skin irritation. Such changes can be brought about through multiple mechanisms (Goh and Soh, 1984; Corsini and Galli, 1998; Welss et al., 2004). At one extreme, widespread killing of the major cellular elements of the skin may occur upon a single exposure to a highly toxic substance. This is referred to as acute skin irritation or as a corrosive effect. More common is irritation resulting from a single or repetitive exposure to mild irritants. These reactions may have a component of cytotoxic cell injury, but the pathophysiology may also reflect non-lethal changes in cell physiology. Agents that cause epidermal hyperplasia and hyperkeratosis or other manifestations of altered differentiation often have an associated irritant dermatitis (Welss et al., 2004). In addition to these reactions that result from direct effects on keratinocytes and fibroblasts are reactions that engage the adaptive immune system as part of the pathophysiology (Goh and Soh, 1984; York et al., 1996; Coquette et al., 2003). Given this multiplicity of mechanisms, and the possibility that certain agents may be allergenic as well as directly irritating (Basketter et al., 2004), it is not surprising that no single test is capable of providing a reliable indicator of risk.

To date, in vivo assays have been used to assess the potential hazard of skin-contact agents. Patch-testing in humans has the potential to be the most reliable since the multiple read-outs (visual observation, color change, itchiness, transepidermal water loss, etc.) that are used as indicators constitute actual skin irritation. Most agents that come into contact with human skin, including the irritants and sensitizers examined here, have been examined in this manner. There appears to be a good relationship between concentrations that give positive results in human patch test assays with concentrations that elicited a response in the culture models examined here. For example, RA and benzalkonium chloride are positive in most individuals tested in the 0.02–0.2% range while SLS gives a positive response in most individuals tested at 1–2% (Wilhelm et al., 1989; Berardesca et al., 1990; Fisher et al., 1991; Geier et al., 2003; Basketter et al., 2004; Jacobs et al., 2004; Molander et al., 2004; Marrakchi and Maibach 2006).

With some of the contact sensitizers, concentrations of up to 5% are routinely used (Hinson et al., 1991; Anderson et al., 2001). Patch testing has also been used in an effort to distinguish irritant and allergic responses. In general, irritant responses arise more rapidly and often subside whereas allergic responses tend to arise more slowly and persist (Jacobs et al., 2004; Welss et al., 2004). Another difference is that irritant responses tend to be dose-dependent. In contrast, once an allergic response has developed, lower concentrations often elicit the same response. Overall, the patch test provides reliable information on irritancy but, for obvious reasons, it is not useful for early-stage testing. Further, it does not lend itself to mechanistic studies.

In vivo testing for skin irritation in animals has been used for years as a way to assess contact hazards (Draize et al., 1944). While this approach provides reliable information at an early stage in a compound’s development, issues include a lack of complete correlation between findings in animals and those in humans, overall expense and ethical considerations. Also, it is difficult to distinguish contact irritant from contact allergenic responses. On the other hand, the Draize approach, like the patch test in humans, relies heavily on clinical features of irritation. Other in vivo assays including the local lymph node assay (LLNA) in its various forms, the guinea pig maximization test and rodent "ear-swelling" assays have proven useful for detecting irritation and, in some cases at least, for predicting potential for contact sensitization (Basketter and Scholes 1992; Van Och et al., 2001). Even these assays are not completely specific, however, since all of the irritants used here have been shown to give positive responses in one or more of these predictive assays (Basketter et al., 2004, 2006). Furthermore, as with other in vivo assays, expense and ethical considerations must be taken into account.

Given the limitation of in vivo testing (especially for hazard assessment at an early stage of a product’s development), it is not surprising that a number of in vitro approaches have been or are being developed as replacements. Epidermal-equivalent and skin-equivalent (epidermis and dermis) cultures have been utilized in conjunction with a variety of biomarkers as a way to assess risk (Ponec and Kempenaar, 1995; Botham et al., 1998; Boelsma et al., 2000; Ponec et al., 2002). While some success has been achieved to date, development of alternative in vitro approaches is still ongoing. The studies described here suggest that a combination of human keratinocytes and fibroblasts in monolayer culture and intact human skin in organ culture may provide information that can be used as an initial screen for skin irritation potential.

Using a series of agents that act directly on the major cellular elements of the skin (i.e., contact irritants) and a panel of contact sensitizers, we observed a variety of responses that appear to distinguish the 2 groups. As a group, the irritants inhibited keratinocyte and fibroblast growth (and were overtly cytotoxic) at concentrations far lower than those needed for growth inhibition with the contact sensitizers. Concomitantly, the same contact irritants produced changes in organ-cultured skin (epidermal hyperplasia/epidermal thickening, abnormal differentiation and necrosis) while the contact sensitizers were virtually without effect. Likewise, we observed changes in the profile of secreted products with the irritants that were not observed with the contact sensitizers.

Although the contact sensitizers were much less stimulatory than the irritants in a variety of assays and required higher concentrations for suppression of keratinocyte and fibroblast proliferation, organ culture fluids from tissue treated with either contact irritants or contact sensitizers elaborated two pro-inflammatory cytokines (IL-6 and IL-8). The 2 cytokines could be detected in control skin organ cultures, and levels of both were increased in the presence of the two contact-sensitizers examined. These data are consistent with previous findings from epidermal-equivalent or skin-equivalent cultures (Ponec and Kempenaar, 1995; Boxman et al., 1996; Corsini and Galli, 1998; Bernhofer et al., 1999a, 1999b; Coquette et al., 2003). While in our small sample of test reagents we found no definitive differences between the contact irritants and contact sensitizers (as groups), the finding that the contact sensitizers did, in fact, raise the levels of the 2 cytokines is significant in that it shows that these agents are not inert in the ex vivo assays utilized here.

Increased cytokine elaboration in organ culture is of interest in that it suggests local production. While it is impossible to have a complete inflammatory response in an isolated tissue piece, these data suggest that at least some of the initiating events following exposure to irritants of one type or another originate in the skin itself. Elaboration of pro-inflammatory cytokines in the epidermis could promote local inflammation (and skin irritation) through the numerous events occurring downstream of the cytokines (Mantovani, 1999). While it is not likely that assessing only two cytokines will be sufficient to establish irritancy or to completely define mechanisms, assessing a panel of cytokines may provide a clear indication of a chemical’s ability to cause irritation, at least in a subset of individuals.

As with all predictive assays, there are issues that need to be addressed with the human skin approach described here. Adult human skin may not be readily available. While we used skin from adult volunteers, others have used discarded skin following surgeries (Lehe et al., 2003). Furthermore, we have demonstrated that neonatal human foreskin can be maintained in organ culture (Varani et al., 1994). Tissue obtained at circumcision would, presumably, be more readily available. Another possibility is rodent skin. While skin from rats and mice can be maintained in organ culture (Varani et al., 2002), the structure of rodent skin is very different from that of humans, which could make interpretation of results difficult. Skin from other animals is also a possibility. Pig skin is very similar to human skin and pig skin has been successfully maintained in organ culture (Ghazizadeh et al., 1998). While the use of either rodent skin or skin from another species does not fit the definition of "animal-free testing," it would greatly reduce the number of animals used.

Another issue is variability. With regard to the cytokine measurements in particular, but with all of the endpoints to some extent, the variability among tissue samples was higher in the presence of the various irritants and sensitizers than it was in controls (either intra-subject or between subjects). The high variability, thus, appears to reflect a differential response to the eliciting agent among individuals rather than differences from biopsy to biopsy within a given donor, or inherent (basal) differences among donors. The high variability, while making statistical analysis difficult (and larger sample size necessary), may be important in its own right. Similar variability is observed in patch-testing (Wilhelm et al., 1989; Fisher et al., 1991; Marrakchi and Maibach, 2006) and in other in vivo risk-assessment assays. It may, ultimately, be possible to use responses seen in organ culture to estimate the percentage of individuals who are likely to have an adverse event to a given agent or as a predictor of who will or will not develop a response to a given agent.

In summary, a wide variety of chemical agents produce irritation when applied topically to the skin. A similar cluster of clinical features can be produced through multiple diverse pathways. Given this multiplicity of mechanisms, it is not surprising that no single profile of cellular, histological or biochemical parameters characterizes all contact irritants or contact sensitizers. Nevertheless, the data presented here suggest that a combination of keratinocytes and fibroblasts in monolayer culture and intact skin in organ culture may provide information that can be used as an initial screen for irritation. The data presented here demonstrate that agents that act directly on the major cellular elements of the skin produce a variety of effects that are not seen with contact sensitizers. While the contact sensitizers used in the present studies were inert in a variety of bioassays, these agents were capable of stimulating production of pro-inflammatory cytokines. While these data need to be verified with a larger panel of agents, our findings suggest that it will, ultimately, be possible to use a combination of cell culture and organ culture as an initial screen for irritant potential, and to help distinguish among potential mechanisms.

	References

TOP Abstract Introduction Materials and Methods Results Discussion References

 

• Anderson, KE, Johansen, JD, Bruze, M, Frosch, PJ, Goossens, A, Lepoittevin, J-P, Rastogi, S, While, I, & Menne, T. (2001). The time-dose-response relationship for elicitation of contact dermatitis in isoeugenol allergic individuals. Toxicol Appl Pharmacol, 170, 166-71[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Basketter, DA, & Scholes, EW. (1992). Comparison of the local lymph node assay with the guinea pig maximization test for the detection of a range of contact allergens. Food Chem Toxicol, 30, 65-9[ISI][Medline] [Order article via Infotrieve]
• Basketter, DA, Marriott, M, Gilmour, NJ, & White, IR. (2004). Strong irritants masquerading as skin allergens: the case of benazlkonium chloride. Contact Dermatitis, 50, 213-7[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Basketter, DA, Andersen, KE, Liden, C, Van Loveren, H, Bowan, A, Kimber, I, Alanko, K, & Berggren, E. (2005). Evaluation of the skin sensitizing potency of chemicals by using the existing methods and considerations of relevance for elicitation. Contact Dermatitis, 52, 39-43[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Basketter, DA, McFadden, J, Evans, P, Andersen, KE, & Jowsey, I. (2006). Identification and classification of skin sensitizers: identifying false positives and false negatives. Contact Dermatitis, 55, 268-73[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Berardesca, E, Fideli, D, Gabba, P, Cespa, M, Rabbiosi, G, & Maibach, HI. (1990). Ranking of surfactant skin irritancy in vivo in man using the plastic occlusion stress test (POST). Contact Dermatitis, 23, 1-5[Medline] [Order article via Infotrieve]
• Bernhofer, LP, Seiberg, M, & Martin, KM. (1999a). The influence of the response of skin equivalent systems to topically applied consumer products by epithelial mesenchymal interaction. Toxicol In Vitro, 13, 219-29[CrossRef][ISI]
• Bernhofer, LP, Barkovic, S, Appa, Y, & Martin, KM. (1999b). IL-1 and IL-1 receptor antagonist secretion from epidermal equivalents and the prediction of the irritation potential of mild soaps and surfactant-based consumer products. Toxicol In Vitro, 13, 231-239[CrossRef][ISI]
• Boelsma, E, Gibbs, S, Faller, C, & Ponec, M. (2000). Characterization and comparison of reconstructed skin models: morphological and immunohistochemical evaluation. Acta Dermato Venereol, 80, 82-8[ISI][Medline] [Order article via Infotrieve]
• Botham, PA, Earl, LK, Fentem, JH, Rouguet, R, & van de Sandt, JJM. (1998). Alternative methods for skin irritation testing. ATLA, 26, 195-211
• Botham, PA. (2004). The validation of in vitro methods for skin irritation. Toxicol Lett, 149, 387-90[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Boukamp, P, Petrussevska, RT, Breitkreutz, D, Hornung, J, Markham, A, & Fusenig, NE. (1998). Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol, 106, 761-71[CrossRef]
• Boxman, IL, Ruwhof, C, Boerman, OC, Lowik, CW, & Ponec, M. (1996). Role of fibroblasts in the regulation of proinflammatory interleukin IL-1, IL-6 and IL-8 levels induced by keratinocyte-derived IL-1. Arch Dermatol Res, 288, 391-8[ISI][Medline] [Order article via Infotrieve]
• Coquette, A, Berna, N, Vandenbosch, A, Rosdy, M, De Wever, B, & Poumay, Y. (2003). Analysis of interleukin-1 (IL-1) and interleukin-8 (IL-8) expression and release in in vitro reconstructed human epidermis for the prediction of in vivo skin irritation and/or sensitization. Toxicol In Vitro, 17, 311-21[ISI][Medline] [Order article via Infotrieve]
• Corsini, E, & Galli, CL. (1998). Cytokines and irritant contact dermatitis. Toxicol Lett, 102, 277-82[CrossRef][Medline] [Order article via Infotrieve]
• Draize, JH, Woodard, G, & Calvery, HO. (1944). Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J Pharmacol Exp Therap, 82, 377-90[Free Full Text]
• Fisher, GJ, Esmann, J, Griffiths, CEM, Talwar, HA, Duell, EA, Hammerberg, C, Elder, JT, Karaban, GD, Nickoloff, BJ, Cooper, KD, & Voorhees, JJ. (1991). Cellular, immunological and biochemical characterization of topical retinoic acid-treated human skin. J Invest Dermatol, 96, 699-707[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Geier, J, Uter, W, Pirker, C, & Frosch, PJ. (2003). Patch testing with the irritant sodium lauryl sulfate (SLS) is useful in interpreting weak reactions to contact allergens as allergic or irritant. Contact Dermatitis, 48, 99-107[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Ghazizadeh, S, Harrington, R, Garfield, J, & Taichman, LB. (1998). Retrovirus-mediated transduction of porcine keratinocytes in organ culture. J. Invest. Dermatol, 111, 492-6
• Goh, CL, & Soh, SD. (1984). Occupational dermatoses in Singapore. Contact Dermatitis, 11, 217-23
• Hinson, M, Bruze, M, & Christensen, OB. (1991). The significance of previous allergic contact dermatitis for elicitation of delayed hypersensitivity to nickel. Contact Dermatitis, 34, 414-8[CrossRef]
• Jacobs, JJL, Lehe, C, Cammans, KDA, Das, PK, & Elliott, GR. (2002). An in vitro model for detecting skin irritants: methyl green-pyronine staining of human skin explant cultures. Toxicol In Vitro, 16, 581-8[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Jacobs, JJL, Lehe, CL, Cammans, KDA, Das, PK, & Elliott, GR. (2004). Assessment of contact allergens by dissociation of irritant and sensitizing properties. Toxicol In Vitro, 18, 681-90[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Lehe, CL, Jacobs, JJL, Elliott, GR, & Das, PK. (2003). A two-centre evaluation of the human organotypic skin explant culture model for screening contact allergens. ATLA, 31, 553-61[Medline] [Order article via Infotrieve]
• Mantovani, A. (1999). The chemokine system: redundancy for robust outputs. Immunol Today, 20, 620-33
• Marrakchi, S, & Maibach, HI. (2006). Sodium lauryl sulfate-induced irritation in the human face: Regional and age-related differences. Skin Pharmacol Physiol, 19, 177-80
• Molander, G, Petman, L, Kannas, L, & Lauerma, AI. (2004). Single doses of local betamethasone do not suppress allergic patch test reactions to nickel sulfate. Contact Dermatitis, 50, 218-21[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Nixon, GA, Tyson, CA, & Wertz, WC. (1975). Interspecies comparisons of skin irritancy. Toxicol Appl Pharmacol, 31, 481-90[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Ponec, M, & Kempenaar, J. (1995). Use of human skin recombinants as an in vitro model for testing the irritation potential of cutaneous irritants. Skin Pharmacol, 8, 49-59[ISI][Medline] [Order article via Infotrieve]
• Ponec, M, Boelsma, E, Gibbs, S, & Mommaas, M. (2002). Characterization of reconstructed skin models. Skin Pharmacol Appl Skin Physiol, 15, 4-17[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Rittié, L, Varani, J, Kang, S, Fisher, GJ, & Voorhees, JJ. (2006). Retinoid-induced epidermal hyperplasia is mediated by epidermal growth factor receptor activation via specific induction of its ligands heparin binding-EGF and amphiregulin in human skin in vivo. J Invest Dermatol, 126, 732-9[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Van Och, FMM, Vandebriel, RJ, Prinsen, MK, de Jong, Wh, Slob, W, & van Loveren, H. (2001). Comparison of dose-responses of contact allergens using the guinea pig maximisation test and the local lymph node assay. Toxicology, 167, 207-15[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Varani, J, Taylor, CG, Dame, M, & Ward, PA. (1992). Human umbilical vein endothelial cell killing by activated neutrophils: Loss of sensitivity to injury is accompanied by decreased iron content during in vitro culture and is restored with exogenous iron. Lab Invest, 66, 708-14[ISI][Medline] [Order article via Infotrieve]
• Varani, J, Fligiel, SE, Schuger, L, Perone, P, Inman, D, Griffiths, CEM, & Voorhees, JJ. (1993). Effects of all-trans retinoic acid and Ca⁺⁺ on human skin in organ culture. Am J Pathol, 142, 189-98[Abstract]
• Varani, J, Perone, P, Griffiths, CEM, Inman, DR, Fligiel, SEG, & Voorhees, JJ. (1994). All-trans retinoic acid (ra) stimulates events in organ-cultured human skin that underlie repair. Adult skin from sun-protected and sun-exposed sites responds in an identical manner to ra while neonatal foreskin responds differently. J Clin Invest, 94, 1747-56[ISI][Medline] [Order article via Infotrieve]
• Varani, J, Warner, RL, Gharaee-Kermani, M, Phan, SH, Kang, S, Chung, JH, Wang, ZQ, Datta, SC, Fisher, GJ, & Voorhees, JJ. (2000). Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin. J Invest Dermatol, 114, 480-6[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Varani, J, Perone, P, Merfert, MG, Moon, SE, Larkin, D, & Stevens, MJ. (2002). All-trans retinoic acid improves structure and function of diabetic rat skin in organ culture. Diabetes, 51, 3510-6[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Welss, T, Basketter, DA, & Schroder, KR. (2004). In vitro skin irritation: facts and future. State of the art review of mechanisms and models. Toxicol In Vitro, 18, 231-43[CrossRef][ISI][Medline] [Order article via Infotrieve]
• Wilhelm, KP, Surber, C, & Maibach, HI. (1989). Quantification of sodium lauryl sulfate irritant dermatitis in man: Comparison of four techniques: skin color reflectance, tranepidermal water loss, laser Doppler flow measurement and visual scores. Arch Dermatol Res, 281, 293-5[CrossRef][ISI][Medline] [Order article via Infotrieve]
• York, M, Griffiths, HA, Whittle, E, & Basketter, DA. (1996). Evaluation of a human patch test for the identification and classification of skin irritation potential. Contact Dermatitis, 34, 204-12[CrossRef][ISI][Medline] [Order article via Infotrieve]

CiteULike

Connotea

Del.icio.us

Digg

Reddit

Technorati

This Article Abstract Free Full Text (Free PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Varani, J. Articles by Dunstan, R. Search for Related Content PubMed PubMed Citation Articles by Varani, J. Articles by Dunstan, R. Social Bookmarking                   What's this?