Upstream eventCovalent Binding, Protein
Key Event Relationship Overview
AOPs Referencing Relationship
Life Stage Applicability
Key Event Relationship Description
Haptens can also react with cell surface proteins and activate response pathways in keratinocytes (see ). Uptake of the hapten by keratinocytes activates multiple events, including the release of pro-inflammatory cytokines and the induction of cyto-protective cellular pathways. Activation of the pro-inflammatory cytokine IL-18 results from cleavage of inactive IL-18 precursor protein by inflammasome-associated caspase-1. Sensitizers can activate the inflammasome (;) and in so doing induce IL-18 production. Intracellular Nodlike receptors (NLR) contain sensors for a number of cellular insults. Upon activation (by a currently unknown mechanism), NLRs oligomerise form molecular complexes (i.e. inflammasomes) that are involved in the activation of inflammatory-associated caspases, including caspase-1. Inductions of intracellular levels of IL-18 exhibit responses upon exposure to sensitizers which can be used to establish potency.
Keratinocyte exposure to sensitizers also results in induction of antioxidant/electrophile response element ARE/EpRE-dependent pathways. Briefly, reactive chemicals bind to Keap1 (Kelch-like ECH-associates protein 1) that normally inhibit the nuclear erythroid 2-related factor 2 (Nrf2). Released Nrf2 interacts with other nuclear proteins and binds to and activates ARE/EpREdependent pathways, including the cytoprotective genes NADPH-quinone oxidoreductase 1 (NQ01) and glutathione S-transferase (GSHST), among others (;).
Evidence Supporting this KER
The in chemico, in vitro, and in vivo experimental evidence is logical and consistent with the mechanistic plausibility proposed by covalent reactions based on the protein binding theory (; ; ). In selected cases, (e.g. 1-chloro-2,4-dinitrobenzenes) where the same compound has been examined in a variety of assays (see Annex 1 of ), the coherence and consistency of the experimental data is excellent. Alternative mechanism that logically present themselves and the extent to which they may distract from the postulated AOP. It should be noted that alternative mechanisms of action, if supported, require a separate AOP. While covalent reactions with thiol groups and to a lesser extent amino groups, are clearly supported by the proposed AOP, reactions targeting other nucleophiles may or may not be supported by the proposed AOP. Limited data on chemical reactivity shows that two competing reactions are possible, the faster reaction dominates. However, this has yet to be proven in vitro or in vivo.
Uncertainties and Inconsistencies
Uncertainties include the structural and physicochemical
cut-offs between theoretical and measured reactivity (), the
significance of the preferred amino acid target (e.g., cysteine versus lysine) (OECD, 2011b), the
significance of Th1 or type 1 (IFN-γ) versus Th2 or type 2 (IL-2, IL-4, IL-13) cytokine secretion
profiles (), and sensitisation measurements in different in vivo models.
Inconsistencies within the reported data are seen. There are differences between in vitro
responses for highly similar chemicals (see;). There
are differences within and between in vivo test results for highly similar chemicals (see Annex C
of the European Centre for Ecotoxicological and Toxicological Chemicals, 2010). Highly
hydrophobic chemicals, which are in vivo sensitizers, are not active in aquatic-based in chemico
or in vitro assays. The specific nature of the relationship between irritation and sensitisation has
yet to be elucidated.
Data gaps: Based on the more than 50 chemical reactions associated with covalent binding to thiol or primary amine moieties in vitro data for keratinocyte, dendritic cell, and T-cell assays, as well as in vivo sensitisation data, is incomplete in that it does not cover the chemical spaces associated with many of these chemical reactions; in chemico data is also incomplete, especially for reactions that favour amino acid targets other than cysteine.
Quantitative Understanding of the Linkage
Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?
An in vitro reporter assay based on activation via the ARE/EpRE response element has been shown to be responsive to known sensitizers in HaCaT keratinocytes. Expression of ARE/EpRE-dependent genes and other cytoprotective genes (including CYP1A1, MT1 and MT2) in HaCaT cells are part of a proprietary in vitro battery approach to determining sensitisation potency (). Both the Natsch and McKim groups have shown that this signalling pathway responds in a quantitative fashion, which is related to LLNA potency (e.g. strong, moderate, and weak).
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
While in vivo testing focuses on selected mammals including man, the key events for this AOP appear to be conserved across mammals. With exceptions, there is agreement between sensitizers initiated by covalent binding to proteins and non-sensitizers tested in mice, guinea-pigs, and humans; this is especially the case for extreme and strong sensitizers but lesser so for weak and non-sensitizers. One problem is that earlier results, especially with the guinea-pig, were not dose-response experiments. Chemical reactivity data show very good concordance of dose-response relationships regardless of the method. In general, available data from in vitro assays are fragmentary and often qualitative (i.e., yes/no).
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