ToxSci Advance Access originally published online on February 16, 2009
Toxicological Sciences 2009 108(2):401-411; doi:10.1093/toxsci/kfp030
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Reactivity Profiling: Covalent Modification of Single Nucleophile Peptides for Skin Sensitization Risk Assessment
Safety and Environmental Assurance Centre, Unilever, Colworth Science Park, Sharnbrook, Bedford MK44 1LQ, UK
1 To whom correspondence should be addressed at Safety and Environmental Assurance Centre, Unilever Colworth, Sharnbrook, Bedford MK44 1LQ, UK. Fax: +44-1234-264-744. E-mail: maja.aleksic{at}unilever.com.
Received December 22, 2008; accepted February 7, 2009
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Abstract |
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The molecular basis of chemical allergy is rooted in the ability of an allergen (hapten) to modify endogenous proteins. This mechanistic understanding aided development of screening assays which generate reproducible quantitative and qualitative reactivity data. Such assays use model peptides with a limited number and type of protein nucleophiles, and the data does not reflect the specificity, variety, and complexity of hapten interactions with multiple nucleophiles. Building on these developments, we extended the standardized approach to maximize the type and the amount of information that can be derived from an in chemico assay. We used a panel of six single nucleophile peptides and individually optimized the incubation conditions to favor chemical modification. Employing liquid chromatography tandem mass spectrometry (LC-MS/MS) technique, we simultaneously obtained multiple quantitative and qualitative measurements (% peptide depletion, adducts formation, and peptide dimerization for Cys-containing peptide). Using these methods, we obtained reactivity data for 36 chemicals of known skin sensitizing potency. By optimizing incubation conditions, we ensured detection of all reactive chemicals. We explored the LC-MS/MS approach to generate kinetic data for 10 chemicals allowing further characterization of reactivity and a potentially more robust quantitative reactivity descriptor. Our ultimate aim is to integrate this dataset with available physicochemical data and outputs from other predictive assays, all addressing different key steps in the induction of sensitization, to help us make decisions about the safe use of chemicals without using animal tests. The epidermal protein target sites, modification of which may be immunogenic and lead to induction of skin sensitization, are currently unknown. Increasing the understanding of this process may help further refine in chemico reactivity assays as well as aid the interpretation of the reactivity data.
Key Words: skin sensitization; covalent binding; model peptide; mass spectrometry; in chemico; hapten.
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