API

Relationship: 376

Title

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Covalent Binding, Protein leads to Response, Keratinocytes

Upstream event

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Covalent Binding, Protein

Downstream event

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Response, Keratinocytes

Key Event Relationship Overview

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AOPs Referencing Relationship

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Taxonomic Applicability

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Sex Applicability

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Life Stage Applicability

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How Does This Key Event Relationship Work

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Haptens can also react with cell surface proteins and activate response pathways in keratinocytes (see [1]). 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[2]. Sensitizers can activate the inflammasome ([3];[4]) 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[5].

Keratinocyte exposure to sensitizers also results in induction of antioxidant/electrophile response element ARE/EpRE-dependent pathways[6]. 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 ([6];[7]).

Weight of Evidence

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Biological Plausibility

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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 ([8]; [9]; [10]). 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 [11]), 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.

Empirical Support for Linkage

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Uncertainties or Inconsistencies

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Uncertainties include the structural and physicochemical cut-offs between theoretical and measured reactivity ([12]), 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 ([13]), 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[6];[14]). 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[15] 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

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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[16]. 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 ([14]). 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).

Evidence Supporting Taxonomic Applicability

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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).

References

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  1. Weltzien H, Corsini E, Gibbs S, Lindstedt M, Borrebaeck C, Budde P, Schulz-Knappe P, Thierse HJ, Martin S, Roggen, E. 2009. Safe cosmetics without animal testing? Contributions of the EU Project Sens-it-iv. J. für Verbraucherschutz und Lebensmittelsicherheit. 4: 41-48.
  2. Martinon F, Mayor A, Tschopp J. 2009. The inflammasomes: guardians of the body. Ann. Rev. Immunol. 27: 229-265.
  3. Sutterwala FS, Ogura Y, Szczepanik M, Lara-Tejero M, Lichtenberger GS, Grant EP, Bertin J, Coyle AJ, Galán JE, Askenase PW, Flavell RA. 2006. Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity 24: 317-327.
  4. Watanabe H, Gaide O, Pétrilli V, Martinon F, Contassot E, Roques S, Kummer JA, Tschopp J, French LE. 2007. Activation of the IL-1beta-processing inflammasome is involved in contact hypersensitivity. J. Invest. Dermatol. 127: 1956-1963.
  5. Van Och FMM, Van Loveren H, Van Wolfswinkel JC, Machielsen AJC, Vandebriel RJ. 2005. Assessment of potency of allergenic activity of low molecular weight compounds based on IL-1α and IL-18 production by a murine and human keratinocyte cell line. Toxicology 210: 95-109.
  6. 6.0 6.1 6.2 Natsch A and Emter R. 2008. Skin sensitizers induce antioxidant response element dependent genes: Application to the in vitro testing of the sensitisation potential of chemicals. Toxicol. Sci. 102: 110-119
  7. Ade N, Leon F, Pallardy M, Pfeiffer JL, Kerdine-Romer S, Tissier MH, Bonnet PA, Fabre I Ourlin JC. 2009. HMOX1 and NQO1 genes are upregulated in response to contact sensitizers in dendritic cells and THP-1 cell line: role of the Keap1/Nrf2 pathway. Toxicol. Sci. 107: 451-460.
  8. Gerberick F, Aleksic M, Basketter D, Casati S, Karlberg AT, Kern P, Kimber I, Lepoittevin JP, Natsch A, Ovigne JM, Rovida C, Sakaguchi H and Schultz T 2008. Chemical reactivity measurement and the predictive identification of skin sensitisers. Altern. Lab. Anim.36: 215-242.
  9. Karlberg AT, Bergström MA, Börje A, Luthman, K, Nilsson JL. 2008. Allergic contact dermatitis- formation, structural requirements, and reactivity of skin sensitizers. Chem. Res. Toxicol. 21: 53-69.
  10. Adler S, BasketterD, Creton S, Pelkonen O, van Benthem J, Zuang V, Ejner-Andersen K, Angers- Loustau A, Aptula A, Bal-Price A, Benfenati E, Bernauer U, Bessems J, Bois FY, Boobis A, Brandon E, Bremer S, Broschard T, Casati S Coecke S Corvi R, Cronin M, Daston G, Dekant W, Felter S, Grignard E, Gundert-Remy U, Heinonen T, Kimber I, Kleinjans J, Komulainen H, Kreiling R, Kreysa J, Batista Leite S, Loizou G, Maxwell G, Mazzatorta P, Munn S, Pfuhler S, Phrakonkham P, Piersma A, Poth A, Prieto P, Repetto G, Rogiers V, Schoeters G, Schwarz M, Serafimova R, Tahti H, Testai E, van Delft J, van Loveren H, Vinken M, Worth A, Zaldivar JM. 2011. Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010. Arch. Toxicol. 85: 367-485.
  11. OECD (2012a) The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins. Part 1: Scientific Evidence. Series on Testing and Assessment No. 168.
  12. Schwöbel JAH, Koleva YK, Bajot F, Enoch SJ, Hewitt M, Madden JC, Roberts DW, Schultz TW, Cronin MTD. 2011. Measurement and estimation of electrophilic reactivity for predictive toxicology. Chem. Rev. 111: 2562-2596
  13. Hopkins JE, Naisbitt DJ, Kitteringham NR, Dearman RJ, Kimber I, Park BK. 2005. Selective haptenation of cellular or extracellular proteins by chemical allergens: Association with cytokine polarization. Chem. Res. Toxicol. 18: 375-381
  14. 14.0 14.1 McKim JM Jr, Keller DJ III, Gorski JR. 2010. A new in vitro method for identifying chemical sensitizers combining peptide binding with ARE/EpRE-mediated gene expression in human skin cells. Cutan. Ocul. Toxicol. 29: 171-192.
  15. OECD 2011b. Report of the Expert Consultation on Scientific and Regulatory Evaluation of Organic Chemistry-based Structural Alerts for the Identification of Protein-binding Chemicals. OECD Environment, Health and Safety Publications Series on Testing and Assessment No. 139. ENV/JM/MONO(2011)9.
  16. Emter R, Ellis G, Natsch A. 2010. Performance of a novel keratinocyte-based reporter cell line in screen skin sensitizers in vitro. Toxicol. Appl. Pharmacol. 245: 281-290.