Pyridoxylamine reactivity kinetics as an amine based nucleophile for screening electrophilic dermal sensitizers

Abstract

Chemical allergens bind directly, or after metabolic or abiotic activation, to endogenous proteins to become allergenic. Assessment of this initial binding has been suggested as a target for development of assays to screen chemicals for their allergenic potential. Recently we reported a nitrobenzenethiol (NBT) based method for screening thiol reactive skin sensitizers, however, amine selective sensitizers are not detected by this assay. In the present study we describe an amine (pyridoxylamine (PDA)) based kinetic assay to complement the NBT assay for identification of amine-selective and non-selective skin sensitizers. UV-Vis spectrophotometry and fluorescence were used to measure PDA reactivity for 57 chemicals including anhydrides, aldehydes, and quinones where reaction rates ranged from 116 to 6.2 × 10−6 M−1 s−1 for extreme to weak sensitizers, respectively. No reactivity towards PDA was observed with the thiol-selective sensitizers, non-sensitizers and prohaptens. The PDA rate constants correlated significantly with their respective murine local lymph node assay (LLNA) threshold EC3 values (R2 = 0.76). The use of PDA serves as a simple, inexpensive amine based method that shows promise as a preliminary screening tool for electrophilic, amine-selective skin sensitizers.

Mechanistic understanding of molecular initiating events (MIEs) using NMR spectroscopy

“…The mechanistic and reactivity data presented here are essentially consistent with previously published data from peptide reactivity assays 30,[33][34][35][36][37]39 and hence demonstrate that the technique can be used to support the study of skin sensitization AOP chemicals within TT21C 1 approaches. Peptide reactivity assays are generally run over 24 h with the electrophile in excess, hence they differ from the NMR methodology described here in which reactions were generally followed for less than one hour and with excess nucleophile.…”

“…24 The study of haptenation reactions has largely been restricted to the use of nucleophilic protein-mimetic models as the protein target is not always identified. [25][26][27][28][29] Indeed, several approaches for the assessment of chemical reactivity towards proteins have been developed, 22,[30][31][32][33][34][35][36][37][38][39] largely driven by the need to assess chemical reactivity in skin sensitization, although it should be noted that they are equally applicable wherever a covalent mechanism of action is involved in the MIE; they include spectrophotometric assays that monitor changes in UV absorbance and fluorescence 32,33 as well as peptide reactivity assays that measure reactivity of nucleophiles to synthetic peptides using HPLC-UV, MS and MS/MS platforms. 30,[34][35][36][37] These assays do, however, have some limitations which include providing limited mechanistic information and indirect reactivity data that are not suitable for risk modelling (i.e.…”

“…For the thiol reactivity studies, an aqueous/acetonitrile co-solvent was used; a similar solvent system to that used in peptide reactivity 30,[34][35][36][37] and spectrophotometric assays. 32,33 One of the aims of the reactivity studies was to generate reaction rate constants that provide an absolute measure of relative reactivity. Rate constants are independent of reactant concentration and so could be obtained using solution concentrations that allowed rapid acquisition of NMR data from the reactions; it should be noted that the reactant concentrations used were similar to those used in peptide reactivity assays 30,[34][35][36][37] and spectrophotometric assays.…”

“…Rate constants are independent of reactant concentration and so could be obtained using solution concentrations that allowed rapid acquisition of NMR data from the reactions; it should be noted that the reactant concentrations used were similar to those used in peptide reactivity assays 30,[34][35][36][37] and spectrophotometric assays. 32,33 Data for each nucleophile were obtained under standardized conditions of reaction environment; this being a prerequisite for obtaining reaction rate constant data to be used comparatively and predictively.…”

Reactive Carbonyl Species Scavengers—Novel Therapeutic Approaches for Chronic Diseases

“…PM showed increased reactivity compared to lysine for MGO, but this enhanced reactivity was at least an order of magnitude less than what was seen with 1,4-dicarbonyls. Other investigators also found that the reaction rate of PM for compounds that were 1,2-dicarbonyls such as glyoxal, 2,3-butadione, and methyl pyruvate were several orders of magnitude slower at reacting with PM than 1,4-dicarbonyls such o -phthaladehyde and benzoquinone [120]. To reduce RCS modification of ubiquitin by 50%, an 8.8 molar excess of PM is required for HNE, a 9.3 molar excess for MGO, and a 1.31 molar excess is need for MDA [93].…”