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Covalent Binding, Protein leads to Increased proinflammatory mediators
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding||Point of Contact||Author Status||OECD Status|
|Covalent Binding of Low Molecular Weight Organic Chemicals to Proteins leads to Sensitisation (Sensitization) of the Respiratory Tract||adjacent||High||Not Specified||Arthur Author (send email)||Under Development: Contributions and Comments Welcome||Under Development|
Life Stage Applicability
Key Event Relationship Description
Covalent binding to proteins by electrophiles generates haptenated proteins which result in measurable increases in . As such, the induction and/or activation of a variety of proinflammatory mediators is a measurable result of stressors that covalently bind proteins.
Evidence Collection Strategy
Evidence Supporting this KER
Evidence mostly from in vitro studies show that respiratory sensitizers are able, via protein-chemical conjugates, to generate cellular danger signals, including proinflammatory cytokines and oxidative stress.
(Hur et al., 2009) found that toluene diisocyanate (TDI)-human serum albumin (HSA) conjugates increased reactive oxygen species (ROS) production in A549 cells. Exposure of reconstituted three-dimensional (3D) human airway epithelia (MulcilAir™) to respiratory sensitizers trimellitic anhydride (TMA) and methylene diphenyl diisocyanate (MDI) elevated the levels of proinflammatory cytokines and chemokines interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1)/chemokine ligand (CCL)2, growth regulated oncogene-a (GRO-a)/C-X-C motif (CX)CL1, and regulated on activation, normal T cell expressed and secreted (RANTES)/CCL5. (Huang et al., 2013) Similarly, typical respiratory sensitizers caused an elevation of proinflammatory cytokines IL-1a (TMA) and tumor necrosis factor (TNF)-a (glutaraldehyde) in precision-cut lung slices. (Lauenstein et al., 2014)
The significance of the Nrf2-Keap1 pathway in respiratory sensitization is not as extensively studied compared to skin sensitization, but in vitro data for a limited number of respiratory sensitizers showed that these are able to activate Nrf2-dependent genes both in airway and skin epithelium. (Emter et al., 2010, Natsch et al., 2013, Remy et al., 2014) Activation of Nrf2-Keap1 by skin sensitizers has been explained by covalent interaction of cysteine residues on Keap1 with cysteine-reactive chemicals, leading to Nrf2 association and transcriptional activation of genes. It is not fully understood how respiratory sensitizers activate this pathway. Although respiratory sensitizers are more likely to bind to hard nucleophiles such as lysine, (Enoch et al., 2010) in chemico studies show that cysteine binding occurs as well. (Lalko et al., 2011, 2013) Hence, Nrf2 activation may be a direct result of covalent interaction with cysteine residues or an indirect result of GSH depletion and an altered redox balance. The indirect activation of Nrf2-dependent genes was shown in THP-1 cells exposed to acid anhydrides, which had a preference to lysine in the direct peptide reactivity assay (DPRA) (Migdal et al., 2013); however, actual Nrf2 and heme oxygenase-1 proteins accumulated only minimally in the cells.
Uncertainties and Inconsistencies
To elucidate which pathways respiratory sensitizers regulate, in vitro DNA microarray studies were performed in different human lung cell lines exposed to a limited set of respiratory sensitizers. These studies were not able to identify specific molecular pathways that were regulated by respiratory sensitizers. They could identify activation of genes, related to innate immune response. In human alveolar epithelial cells (A549 cell line), for example, genes encoding for TLR2, TNF-a, IL-1 receptor, and cytokine signaling pathways were upregulated by hexamethylene diisocyanate (HDI) and TMA. (Verstraelen et al., 2009) NLRP3 has been demonstrated to be important in respiratory sensitization by proteins, (Besnard et al., 2012) but the involvement in the induction of respiratory sensitization by low-molecular-weight chemicals is unknown. In human keratinocytes, the respiratory sensitizers MDI and TMA failed to elevate intracellular proinflammatory IL-18 levels. (Corsini et al., 2009) Conflicting reports as to whether IL-18 is associated with a Th1 or Th2 immune response hamper interpretation of this result.
Additionally, the canonical phosphatase and tensin homolog (PTEN)-signaling pathway might be relevant for respiratory sensitization. (Verstraelen et al., 2009) This pathway regulates cell survival signaling pathways and plays a protective role in the pathogenesis of asthma. (Kwak et al., 2003) In a mouse model of TDI-induced asthma, the PTEN pathway was shown to play a protective role in asthma pathogenesis, because it was involved in the regulation of IL-17 induction and NF-kB activation. (Kim et al., 2007) A more recent in vitro study showed that the PTEN pathway was not consistently induced by all respiratory sensitizers, since maleic anhydride and 7-aminocephalosporanic acid failed to induce this pathway but another diisocyanate, HDI, did. (Remy et al., 2014)
Known modulating factors
Respiratory sensitizers without intrinsic electrophilic activity have been observed, and this is attributed to in situ generation of electrophilic activity. Pre-haptens and pro-haptens are converted from inactive molecules into active electrophiles by UV light and metabolic enzymes, respectively. (Aptula et al., 2007)
(Taylor et al;, 2020) found that single nucleotide polymorphisms (SNPs) in genese regulating inflammation, calcium regulation and endothelial function, and serine/threonine protein kinsase signaling were associated with differences in plasma and urine levels of 1,6-hexamethylene diisocyanate monomer and 1,6-hexamethylene diisocyanate isocyanurate following occupational exposure.
Quantitative Understanding of the Linkage
Haptenation is essentially instantaneous, and inflammatory responses to haptenated proteins are rapid. As a result, in vitro cytokine/chemokine secretion and redox responses may be quantifiable within minutes to a few hours, but sensitivity and precision vary based on the assay detection method. Haptenated peptides generated in vitro can be quantified after 15 minutes. (Hettick, et al., 2009) Most in vitro cellular assay protocols quantify inflammatory readouts after 24 – 48 hours of exposure.
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
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