ToxSci Advance Access published online on September 12, 2008
Toxicological Sciences, doi:10.1093/toxsci/kfn194
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LC-MS based characterization of the peptide reactivity of chemicals to improve the in vitro prediction of the skin sensitization potential
Givaudan Schweiz AG, Ueberlandstrasse 138, CH-8600 Duebendorf, Switzerland
* corresponding author, andreas.natsch{at}givaudan.com, Tel.: ++41 44 824 21 05, Fax: ++41 824 29 26
Received June 26, 2008; revision received September 1, 2008; accepted September 4, 2008
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Abstract |
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A key step in the skin sensitization process is the formation of a covalent adduct between skin sensitizers and endogenous proteins and/or peptides in the skin. Based on this mechanistic understanding, there is a renewed interest in in vitro assays to determine the reactivity of chemicals towards peptides in order to predict their sensitization potential. A standardized peptide reactivity assay yielded a promising predictivity. This published assay is based on HPLC with UV detection to quantify peptide depletion after incubation with test chemicals. We had observed that peptide depletion may be due to either adduct formation or peptide oxidation. Here we report a modified assay based on both LC-MS analysis and detection of free thiol groups. This approach allows simultaneous determination of (i) peptide depletion, (ii) peptide oxidation (dimerization), (iii) adduct formation and (iv) thiol reactivity, and thus generates a more detailed characterization of the reactivity of a molecule. Highly reactive molecules are further discriminated with a kinetic measure. The assay was validated on 80 chemicals. Peptide depletion could accurately be quantified both with LC-MS detection and depletion of thiol groups. The majority of the moderate/strong/extreme sensitizers formed detectable peptide-adducts, but many sensitizers were also able to catalyze peptide oxidation. Whereas adduct formation was only observed for sensitizers, this oxidation reaction was also observed for two non-sensitizing fragrance aldehydes, indicating that peptide depletion might not always be regarded as sufficient evidence for rating a chemical as a sensitizer. Thus this modified assay gives a more informed view of the peptide reactivity of chemicals to better predict their sensitization potential.
Key Words: Alternative test; skin sensitization; LC-MS; peptide reactivity; adduct formation.
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