Metabolic Epoxidation of an α,β-Unsaturated Oxime Generates Sensitizers of Extreme Potency. Are Nitroso Intermediates Responsible?

Abstract

Metabolic activation of inherently nonprotein-reactive compounds (prohaptens) in the skin can lead to development of contact allergy, a chronic skin disease. The prohapten hypothesis has existed for more than 20 years; yet, detailed knowledge regarding the mechanisms of activation as well as what structural moieties can be transformed to protein-reactive sensitizers is still limited. Today, the consideration of cutaneous bioactivation is important when developing nonanimal-based assays for prediction of contact allergenic activity, as only methods that include skin metabolism are able to detect prohaptens as sensitizers. We have studied the mechanism of activation of the prohapten carvoxime (1), a strongly sensitizing but in itself poorly protein-reactive alpha,beta-unsaturated oxime. alpha,beta-Unsaturated oximes represent a novel class of prohaptens, which previously have never been investigated for potential metabolic activation. To identify reactive metabolites formed from 1, liver microsomal incubations in the presence of glutathione were carried out. Putative reactive metabolites were synthesized, and their allergenic activity, chemical reactivity toward nucleophiles, and ability to elicit a contact allergenic response in animals induced with 1 were assessed. We found that 1 is metabolically activated by epoxidation of the allylic carbon-carbon double bond. The alpha,beta-epoxy oxime metabolites were found to be sensitizers of extreme potency in the local lymph node assay and highly reactive toward nucleophilic amino acids and a model peptide. One of the two diastereomeric epoxy metabolites also elicited an allergic reaction in mice sensitized to 1, in the mouse ear swelling test. Furthermore, this study presents strong indications that the basis of the high reactivity and sensitizing capacity observed for the alpha,beta-unsaturated oximes is related to their ability to form highly reactive nitroso intermediates by tautomerization. To our knowledge, the formation of nitrosoalkenes by oxidative metabolism of alpha,beta-unsaturated oximes has not been shown so far.

Chemical Reactivity Measurement and the Predictive Identification of Skin Sensitisers

“…Few thorough mechanistic investigations on prohaptens in contact allergy have been published up to now, e.g. the studies on the alpha,beta-unsaturated primary alcohol, cinnamic alcohol (Figure 6; 79-81), on alkenes (Figure 7; 82-84), and on oximes (85). However, based on knowledge of xenobiotic bioactivation reactions, clinical observations and/or in vivo and in vitro studies on sensitising capacity and chemical reactivity, numerous skin sensitising pro-haptens can be recognised.…”

The Fate of a Hapten - From the Skin to Modification of Macrophage Migration Inhibitory Factor (MIF) in Lymph Nodes

“…Traditionally, experiments to identify targets for haptens have mainly focused on reactivity toward cysteine and lysine and although most haptens have been shown to be more reactive toward cysteine than lysine, we as well as others have demonstrated that hapten-cysteine adducts are often unstable 7 , 39 – 41 . There are, however, some studies by us and others in which the reactivity of the peptide PHCKRM toward a number of haptens, such as isothiocyanates 7 , epoxides 42 – 44 , oximes 45 , and anhydrides 41 has been investigated. These studies found that the investigated haptens formed stable adducts by covalent binding to the N-terminal proline of PHCKRM.…”

Hydroxylamines and Oximes: Biological Properties and Potential Uses as Therapeutic Agents

“…They can undergo epoxidation that eventually produces reactive nitroso intermediates which act as strong contact sensitizers 140 . Here, the oxime function plays a major role in the chemical activation that results in the reactive nitrosoalkenes.…”

“…It was further pointed by the authors that oximes can be hydrolyzed in vivo to the corresponding carbonyl compounds which are potential allergens. It is of interest to point out in this context that a closely related analog of 46, perillartine (47) 183 , is used in Japan as artificial sweetner 140 , and apparently is devoid of the physiological untoward effects of 46. Perillartine was used as a lead substance for systematic study of numerous combinations of an α,β-unsaturation group and an aldoxime function as sweeteners or bitter molecules 183,184 .…”

“…Biochemical transformations of the −C=N−OH function of synthetic drugs to an NO 132 and to nitroso intermediates140 were recently demonstrated as a new pathway for metabolic activation of oxime-containing molecules. These reactions were recently explored in detail, and may underlie possible biochemical transformations of the −C=N− OH moiety in mammalian tissues, which are likely to be catalyzed by cytochrome P450 and by flavin-containing monooxygenase141 .…”