Elsevier

Toxicology in Vitro

Volume 15, Issues 4–5, August–October 2001, Pages 393-398
Toxicology in Vitro

Gene expression analysis of EpiDerm following exposure to SLS using cDNA microarrays

https://doi.org/10.1016/S0887-2333(01)00042-XGet rights and content

Abstract

There is a need to investigate the mechanistic basis of the human skin irritation response if relevant in vitro test systems for the predictive identification of skin irritation hazards are to be developed. Recent progress in genomics technologies mean that tools for the identification and investigation of important biochemical events in the processes of skin irritation are now available. The aim of this work was to identify genes (for further mechanistic investigation) which may be regulated in response to skin irritation, following exposure of the EpiDerm™ skin model to the known skin irritant sodium lauryl sulphate (SLS). EpiDerm cultures were treated in triplicate with a non-cytotoxic dose of SLS (0.1 mg/ml, as determined by the MTT assay and histological examination) for 15 min, 30 min, 1 h, 2 h, 3 h, 4 h and 24 h. Total RNA was extracted from the pooled EpiDerm cultures and used to probe Atlas™ human arrays (Clontech) covering approximately 3600 genes. Preliminary data indicated an up-regulation at early time points (15–30 min) of a number of genes involved in transportation (e.g. the sodium and chloride dependent taurine transporter) and receptors (e.g. ZAP70 and protocadherin 42 precursor). The gene encoding the UV excision repair protein and other DNA repair genes (e.g. DNA-directed RNA polymerase II) were up-regulated after 1–3 h, along with TGFβ3 and other tumour suppressors, which play a role in cellular development and wound healing. At the later time points of 4–24 h, genes involved in protein translation (e.g. Cathepsin D receptor) and metabolism (e.g. CYP27A) were up-regulated. In addition, a number of genes were down-regulated in response to treatment with SLS, although these followed less of a time dependent pattern. These results indicate the differential regulation of a number of genes in response to treatment with SLS, some of which may provide additional clues to the molecular events underpinning the irritation response to this particular surfactant and possibly to other chemical irritants.

Introduction

Development of relevant in vitro test systems for the predictive identification of skin irritation hazards requires an understanding of the mechanistic basis of the human skin irritation response. Recent progress in genomics technologies means that tools for the identification and investigation of important biochemical events in the processes of skin irritation are now available (e.g. large-scale differential gene expression using microarray hybridisation technology). These tools can be used to identify genes critical in the human skin response following exposure to irritants. It is currently difficult to differentiate between irritant contact dermatitis and allergic contact dermatitis both clinically and histopathologically. However, they are believed to initiate responses via different pathways (Harvell and Maibach, 1994), a period of induction after initiation being required for an immune mediated (i.e. allergic) response but not for irritant responses. A strong allergen may serve as both inductor and elicitor on exposure to skin, making the distinction between irritation and allergy difficult. In practice, time course assessments do not provide a reliable distinction between irritant and allergic reactions because in patch testing, irritants can mimic weak allergens by intensifying the reaction after an initial reading is taken (Rietschel, 1997). Basketter (1998) hypothesised that all chemicals are skin irritants to some degree, it is a matter of low irritant potency and relatively limited skin exposure that makes some substances appear non-irritant. Current methods of identifying skin irritants using monolayer cultures (Gueniche and Ponec, 1993) with IL-1α or lactate dehydrogenase (LDH) activity as endpoints (Cohen et al., 1991, Osborne and Perkins, 1991) do not reliably distinguish between the irritant and allergic response. By using genomics technology thousands of genes can be screened in one experiment to provide a ‘global view’ of genes involved in the skin irritation response. Some of these genes may provide new molecular markers of skin irritation in the future.

The aim of this work was to identify genes for further investigation, which may be regulated in response to skin irritation, following exposure of the EpiDerm skin model to the known skin irritant SLS.

Section snippets

MTT cytotoxicity assay

To ascertain non-cytotoxic doses of the skin irritant SLS (Sigma), the same sample was used in all experiments, an MTT assay was performed. EpiDerm (EPI-200, cultured in the absence of hydrocortisone, supplied by MatTek) were treated in triplicate following equilibration with 20, 10, 1, 0.1 and 0.01 mg/ml SLS for 30 min, 1, 2, 3, 4 and 24 h. MTT solution was prepared as follows: MTT concentrate (provided by MatTek) was diluted with MTT diluent (provided by MatTek) to produce a 1 mg/ml solution.

MTT cytotoxicity assay

EpiDerm cultures were treated with 20, 10, 1, 0.1 and 0.01 mg/ml SLS for 30 min, 1, 2, 3, 4 and 24 h. The results of the MTT assay are shown in Fig. 1. The corresponding EC50 values were: >100 mg/ml after 30 min and 1 h of treatment with SLS, 12.9±3 mg/ml at 2 h, 3.7±0.5 mg/ml at 3 h, 5.2±0.6 mg/ml after 4 h and 2.1±0.9 mg/ml after 24 h. 0.1 mg/ml SLS was chosen as the dose to be used in the microarray experiments as it showed limited cell damage (following histological examination, results not

Discussion

Traditionally the methods used to investigate skin irritation have been performed in vivo using laboratory animals (primarily rabbits), the most notable of which is the Draize rabbit skin irritation test where the test material is applied to the shaved skin of albino rabbits (Draize et al., 1944). This method has the potential to cause discomfort or pain to the animal and therefore the use of alternative test methods is being investigated. The Draize test has also been criticised for a number

References (18)

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