ToxSci Advance Access originally published online on September 11, 2009
Toxicological Sciences 2009 112(1):164-174; doi:10.1093/toxsci/kfp192
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Investigation of Peptide Reactivity of Pro-hapten Skin Sensitizers Using a Peroxidase-Peroxide Oxidation System
* Central Product Safety, Miami Valley Innovation Center, The Procter & Gamble Company, Cincinnati, Ohio 45253
Analytical Global Capability Organization, Mason Business Center, The Procter & Gamble Company, Cincinnati, Ohio 45040
Central Product Safety, Darmstadt Innovation Center, The Procter & Gamble Service GmbH, 64283 Darmstadt, Germany
University of Strasbourg, Laboratoire de Dermatochimie, UMR 7177 Strasbourg, France
1 To whom correspondence should be addressed at Central Product Safety, Miami Valley Innovation Center, The Procter & Gamble Company, P.O. Box 538707, Cincinnati, OH 45253-8707. Fax: (513) 627-0400. E-mail: gerberick.gf{at}pg.com.
Received June 25, 2009; accepted August 4, 2009
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Abstract |
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Skin protein reactivity is a well established key step in the development of skin sensitization. Understanding the relationship between a chemical's ability to react with or modify skin protein and skin sensitization has led to the development of the Direct Peptide Reactivity Assay (DPRA) in our laboratory. A current limitation of the DPRA is that it cannot readily measure the reactivity of pro-hapten chemical sensitizers. Pro-haptens are chemical sensitizers that are not directly reactive and must be bioactivated in vivo to form an electrophilic intermediate(s). Results from this work demonstrate the utility of using horseradish peroxidase and hydrogen peroxide (HRP/P) for assessing the skin sensitization potential of pro-haptens. In comparison with "direct" reactivity assessments without HRP/P, statistically significant increases in peptide depletion for all pro-haptens examined were observed following coincubation with HRP/P. Conversely, the percent peptide depletion for all pre-haptens was equally high (> 40% depletion) with and without HRP/P demonstrating an auto-oxidation pathway. In contrast, peptide depletion for all nonsensitizing chemicals examined was low with and without HRP/P. The optimal HRP/P concentrations, incubation time and optimal peptide:chemical ratio were determined using a sensitive and selective high-performance liquid chromatography tandem mass spectrometry detection method. Dithiothreitol was incorporated to reverse the dimerization of the thiol-containing cysteine peptide nucleophile. This preliminary work shows the potential to incorporate an enzyme-mediated activation step for pro-haptens into an in chemico skin sensitization assay that results in the detection of all types of sensitizers.
Key Words: allergens; alternatives; skin sensitization; peptide reactivity.