Location: Natural Products Utilization ResearchTitle: A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers
|AVONTO, CRISTINA - University Of Mississippi|
|CHTTIBOYINA, AMAR - University Of Mississippi|
|RUA, DIEGO - Food And Drug Administration(FDA)|
|KHAN, IKHLAS - University Of Mississippi|
Submitted to: Journal of Toxicology and Applied Pharmacology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/29/2015
Publication Date: 10/9/2015
Citation: Avonto, C., Chttiboyina, A.G., Rua, D., Khan, I.A. 2015. A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers. Journal of Toxicology and Applied Pharmacology. 289:177-184.
Interpretive Summary: In summary, in the present work we propose the development and validation of an innovative approach to assess the skin sensitization potential of chemicals. The evaluation of skin sensitization potential is an important toxicological aspect in the safety evaluation of chemicals intended for use as topical formulations of drugs, cosmetics, fragrances and other personal-care products. The need of alternative methods has been reinforced by regulatory agencies worldwide, which are pushing toward a replacement of in vivo methods with non-animal alternatives for toxicological screenings. Chemical methods are a useful tool in skin sensitization risk assessment. The rationale behind chemical approaches is based on the hypothesis that skin sensitizers must covalently bind to a nucleophilic biological target in order to elicit an allergic response. So far, only one in chemico method has been validated. In this paper, we propose the development of a faster and sensitive fluorescence-based in chemico alternative. The method has been here validated against a panel of known sensitizers and compared to state-of-the-art assays for sensitivity, accuracy and specificity.
Technical 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 productswere analyzed and a prediction of their sensitization potential is presented for the first time.