Refinement of the Peroxidase Peptide Reactivity Assay and Prediction Model for Assessing Skin Sensitization Potential
| Autor: | G. Frank Gerberick, Cindy A. Ryan, Thomas M Burt, John A. Troutman, Petra S. Kern, Roy L. M. Dobson, Hong Jian Dai, Mike Quijano |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Lysine Peptide 010501 environmental sciences Toxicology Animal Testing Alternatives 01 natural sciences 03 medical and health sciences medicine Animals Reactivity (chemistry) Cysteine Sensitization 0105 earth and related environmental sciences Skin chemistry.chemical_classification Chromatography biology Local lymph node assay Allergens Local Lymph Node Assay 030104 developmental biology medicine.anatomical_structure chemistry Peroxidases Dermatitis Allergic Contact biology.protein Peptides Hapten Haptens Peroxidase |
| Zdroj: | Toxicological sciences : an official journal of the Society of Toxicology. 178(1) |
| ISSN: | 1096-0929 |
| Popis: | A peptide reactivity assay with an activation component was developed for use in screening chemicals for skin sensitization potential. A horseradish peroxidase-hydrogen peroxide (HRP/P) oxidation system was incorporated into the assay for characterizing reactivity of hapten and pre-/prohapten sensitizers. The assay, named the Peroxidase Peptide Reactivity Assay (PPRA) had a predictive accuracy of 83% (relative to the local lymph node assay) with the original protocol and prediction model. However, apparent false positives attributed to cysteine depletion at relatively high chemical concentrations and, for some chemicals expected to react with the −NH2 group of lysine, little to no depletion of the lysine peptide were observed. To improve the PPRA, cysteine peptide reactions with and without HRP/P were modified by increasing the number of test concentrations and refining their range. In addition, removal of DL-dithiothreitol from the reaction without HRP/P increased cysteine depletion and improved detection of reactive aldehydes and thiazolines without compromising the assay’s ability to detect prohaptens. Modification of the lysine reaction mixture by changing the buffer from 0.1 M ammonium acetate buffer (pH 10.2) to 0.1 M phosphate buffer (pH 7.4) and increasing the level of organic solvent from 1% to 25% resulted in increased lysine depletion for known lysine reactive chemicals. Refinement of the prediction model improved the sensitivity, specificity, and accuracy for hazard identification. These changes resulted in significant improvement of the PPRA making it is a reliable method for predicting the skin sensitization potential of all chemicals, including pre-/prohaptens and directly reactive haptens. |
| Databáze: | OpenAIRE |
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