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ToxSci Advance Access published online on July 14, 2004
Toxicological Sciences, doi:10.1093/toxsci/kfh213
Toxicological Sciences © Society of Toxicology 2004; all rights reserved
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Received April 26, 2004
Accepted June 22, 2004

In Vitro Toxicology

Development of a Peptide Reactivity Assay for Screening Contact Allergens

G. Frank Gerberick 1*, Jeff D. Vassallo 1, Ruth E. Bailey 1, Joel G. Chaney 1, Steve W. Morrall 1, Jean-Pierre Lepoittevin 2

1 The Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, OH, 45253-8707, USA
2 Université Louis Pasteur, Laboratorie de Dermatochimie, UMR 7123, Strasbourg, France

* To whom correspondence should be addressed. E-mail: gerberick.gf{at}pg.com.


  Abstract

Allergic contact dermatitis resulting from skin sensitization is a common occupational and environmental health problem. In recent years, the local lymph node assay (LLNA) has emerged as a practical option for assessing the skin sensitization potential of chemicals. In addition to accurate identification of skin sensitizers, the LLNA can also provide a reliable measure of relative sensitization potency; information that is pivotal in successful management of human health risks. However, even with the significant animal welfare benefits provided by the LLNA, there is still interest in the development of non-animal test methods for skin sensitization testing. One characteristic of a chemical allergen is its ability to react with proteins prior to the induction of skin sensitization. The majority of chemical allergens is electrophilic and as such react with nucleophilic amino acids like cysteine or lysine. In order to determine if reactivity correlates with sensitization potential, 38 chemicals representing allergens of different potencies (weak to extreme) and non-sensitizers were evaluated for their ability to react with glutathione or three synthetic peptides containing either cysteine, lysine or histidine. Following a 15 minute reaction time for glutathione or a 24 hour reaction period for the 3 synthetic peptides, the samples were analyzed by HPLC. UV detection was used to monitor the depletion of glutathione or the peptide following reaction. The results demonstrate that a significant correlation (Spearman correlation) exists between allergen potency and the depletion of glutathione (p=0.001), lysine (p=0.025) and cysteine (p=0.020), but not histidine. The peptide with the highest sensitivity was cysteine (80.8%) whereas histidine was the least sensitive (11.5%). The data presented show that measuring peptide reactivity has utility for screening chemicals for their skin sensitization potency and thus potential for reducing our reliance on animal test methods.

Keywords: Allergens; alternatives; skin sensitization; peptide reactivity.
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