title: A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers.
abstract: Skin sensitization is an important toxicological end-point in the risk assessment of chemical allergens. Because of the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace conventional animal tests. Chemical methods are intended to characterize the potential of a sensitizer to induce earlier molecular initiating events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features to covalently bind to the biological target and induce further haptenation processes. Current in chemico assays rely on the quantification of unreacted model nucleophiles after incubation with the candidate sensitizer. In the current study, a new fluorescence-based method, 'HTS-DCYA assay', is proposed. The assay aims at the identification of reactive electrophiles based on their chemical reactivity toward a model fluorescent thiol. The reaction workflow enabled the development of a High Throughput Screening (HTS) method to directly quantify the reaction adducts. The reaction conditions have been optimized to minimize solubility issues, oxidative side reactions and increase the throughput of the assay while minimizing the reaction time, which are common issues with existing methods. Thirty-six chemicals previously classified with LLNA, DPRA or KeratinoSens™ were tested as a proof of concept. Preliminary results gave an estimated 82% accuracy, 78% sensitivity, 90% specificity, comparable to other in chemico methods such as Cys-DPRA. In addition to validated chemicals, six natural products were analyzed and a prediction of their sensitization potential is presented for the first time.
authors.affiliation: National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677, United States.
authors.forename: Cristina
authors.initials: C
authors.lastname: Avonto
authors.affiliation: National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677, United States.
authors.forename: Amar G
authors.initials: AG
authors.lastname: Chittiboyina
authors.affiliation: The Center for Food Safety and Applied Nutrition, US Food and Drug Administration, College Park, MD 20740, United States.
authors.forename: Diego
authors.initials: D
authors.lastname: Rua
authors.affiliation: National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677, United States; Division of Pharmacognosy, Department of BioMolecular Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677, United States. Electronic address: ikhan@olemiss.edu.
authors.forename: Ikhlas A
authors.initials: IA
authors.lastname: Khan
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000633:toxicity*; D000826:Animal Testing Alternatives; D000818:Animals; D002138:Calibration; D003543:Cysteamine / Q000031:analogs & derivatives / Q000737:chemistry*; D003619:Dansyl Compounds / Q000737:chemistry*; D017449:Dermatitis, Allergic Contact / Q000209:etiology*; D005456:Fluorescent Dyes / Q000737:chemistry*; D057166:High-Throughput Screening Assays / Q000592:standards; D006801:Humans; D023441:Local Lymph Node Assay; D012015:Reference Standards; D015203:Reproducibility of Results; D018570:Risk Assessment; D023422:Skin Irritancy Tests / Q000379:methods* / Q000592:standards; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D057188:Workflow
chemical_list: D000485:Allergens; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D003543:Cysteamine
keywords: Electrophiles,; Fluorescence assay,; High throughput screening; In chemico alternative methods,; Skin sensitization,