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Relationship: 88


A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

N/A, Covalent binding to protein, possibly lysine residue leads to Activation, Inflammatory cytokines, chemokines, cytoprotective gene pathways

Upstream event
The causing Key Event (KE) in a Key Event Relationship (KER). More help
Downstream event
The responding Key Event (KE) in a Key Event Relationship (KER). More help

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER.  More help
Term Scientific Term Evidence Link
human Homo sapiens NCBI
mouse Mus musculus NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help
Sex Evidence
Unspecific High

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER.  More help
Term Evidence
All life stages Not Specified

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Covalent binding to proteins by electrophiles generates haptenated proteins which are able to activate cytoprotective pathways and ultimately elicit immune defenses.  For respiratory sensitization, there is evidence to suggest binding to specific lysine residues of serum proteins may be the characteristic initiating event.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings.  Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Evidence mostly from in vitro studies show that respiratory sensitizers are able, via protein-chemical conjugates, to generate cellular danger signals, including induction of oxidative stress and proinflammatory cytokines and chemokines. A link between oxidative stress and the initiation of signal transduction pathways involved in inflammation and allergy has been shown. In the skin, for example, oxidative stress may lead to activation of signal transduction pathways such as NF-kB and p38 MAPK, which leads to the release of cytokines and chemokines.

Biological Plausibility
Addresses the biological rationale for a connection between KEupstream and KEdownstream.  This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured.   More help

Multiple cell types in the lung express the necessary pattern recognition receptors for this KER, including epithelial cells, endothelial cells, macrophages, fibroblasts, and dendritic cells. It is likely that different cell types are involved in the process.

(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.

There is some evidence to support the hypothesis that the binding behavior of respiratory sensitizers is related to the eventual Th2-skewed immune response, with binding to lysine on serum albumin in particular, as well as secretion of type 2 cytokines, being associated with known respiratory sensitizers. (Hopkins et al., 2005) The biological hypothesis that lysine is the primary nucleophile responsible for respiratory sensitization is supported by the preference for harder electrophiles compared with those that cause skin sensitization (lysine is a harder nucleophile than cysteine). (Enoch et al., 2010) This is evidenced by the difference in the coverage of the various mechanistic domains that show typical respiratory sensitizers to be chemicals acting via the acylation and Schiff base mechanisms rather than Michael addition (a significantly important mechanism for skin sensitization). Structure/activity analysis has shown the importance of electrophilicity and protein crosslinking for respiratory sensitization for low-molecular-weight organic chemicals. (Hopkins et al., 2005, Agius et al., 1991, 1994, Seed and Agius, 2010, 2017)

One proposed explanation (Kimber et al., 2018) for the association between peptide selectivity and deviation between respiratory and dermal sensitization is based on the observation that respiratory sensitizers, in a co-culture including both U937 cells and serum, preferentially react with serum proteins such as albumin, which has a high number of lysine residues. This behavior was observed for TMA, fluorescein isothiocyanate (FITC), and dinitrobenzenesulfonyl chloride (DNBSCl). Concordantly, skin sensitizers dinitrochlorobenzene (DNCB), dinitrofluorobenzene (DNFB) preferentially bound to cellular proteins in the same co-culture. (Hopkins et al., 2005) This is corroborated by the observation that serum albumin is a major target protein of the respiratory sensitizer hexahydropthalic anhydride in humans. (Johannesson et al., 2001) Further, this is a reasonable hypothesis for the biological mechanism of deviation between skin and respiratory sensitizers, particularly in the case of dermal exposure, as the distribution of antigen formation of chemical allergens in the in vitro model system segregates with the type (Th1- or Th2-activating) of cytokines secreted from activated lymph node cells in an in vivo mouse model.

Uncertainties and Inconsistencies
Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

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

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references.  More help

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)

Response-response Relationship
Provides sources of data that define the response-response relationships between the KEs.  More help
Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

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. Most in vitro sensitization assay protocols quantify readouts after 24 – 48 hours of exposure.

Known Feedforward/Feedback loops influencing this KER
Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help


List of the literature that was cited for this KER description. More help

AGIUS, R. M., ELTON, R. A., SAWYER, L. & TAYLOR, P. 1994. Occupational asthma and the chemical properties of low molecular weight organic substances. Occup Med (Lond), 44, 34-6.

AGIUS, R. M., NEE, J., MCGOVERN, B. & ROBERTSON, A. 1991. Structure activity hypotheses in occupational asthma caused by low molecular weight substances. Ann Occup Hyg, 35, 129-37.

APTULA, A. O., ROBERTS, D. W. & PEASE, C. K. 2007. Haptens, prohaptens and prehaptens, or electrophiles and proelectrophiles. Contact Dermatitis, 56, 54-56.

BESNARD, A. G., TOGBE, D., COUILLIN, I., TAN, Z., ZHENG, S. G., ERARD, F., LE BERT, M., QUESNIAUX, V. & RYFFEL, B. 2012. Inflammasome-IL-1-Th17 response in allergic lung inflammation. J Mol Cell Biol, 4, 3-10.

CORSINI, E., MITJANS, M., GALBIATI, V., LUCCHI, L., GALLI, C. L. & MARINOVICH, M. 2009. Use of IL-18 production in a human keratinocyte cell line to discriminate contact sensitizers from irritants and low molecular weight respiratory allergens. Toxicol In Vitro, 23, 789-96.

EMTER, R., ELLIS, G. & NATSCH, A. 2010. Performance of a novel keratinocyte-based reporter cell line to screen skin sensitizers in vitro. Toxicol Appl Pharmacol, 245, 281-90.

ENOCH, S. J., ROBERTS, D. W. & CRONIN, M. T. 2010. Mechanistic category formation for the prediction of respiratory sensitization. Chem Res Toxicol, 23, 1547-55.

HUANG, S., WISZNIEWSKI, L., CONSTANT, S. & ROGGEN, E. 2013. Potential of in vitro reconstituted 3D human airway epithelia (MucilAir™) to assess respiratory sensitizers. Toxicol In Vitro, 27, 1151-6.

HUR, G. Y., KIM, S. H., PARK, S. M., YE, Y. M., KIM, C. W., JANG, A. S., PARK, C. S., HONG, C. S. & PARK, H. S. 2009. Tissue transglutaminase can be involved in airway inflammation of toluene diisocyanate-induced occupational asthma. J Clin Immunol, 29, 786-94.

JOHANNESSON, G., ROSQVIST, S., LINDH, C. H., WELINDER, H. & JÖNSSON, B. A. 2001. Serum albumins are the major site for in vivo formation of hapten-carrier protein adducts in plasma from humans and guinea-pigs exposed to type-1 allergy inducing hexahydrophthalic anhydride. Clin Exp Allergy, 31, 1021-30.

KIM, S. R., LEE, K. S., PARK, S. J., MIN, K. H., LEE, K. Y., CHOE, Y. H., LEE, Y. R., KIM, J. S., HONG, S. J. & LEE, Y. C. 2007. PTEN down-regulates IL-17 expression in a murine model of toluene diisocyanate-induced airway disease. J Immunol, 179, 6820-9.

KIMBER, I., POOLE, A. & BASKETTER, D. A. 2018. Skin and respiratory chemical allergy: confluence and divergence in a hybrid adverse outcome pathway. Toxicol Res (Camb), 7, 586-605.

KWAK, Y. G., SONG, C. H., YI, H. K., HWANG, P. H., KIM, J. S., LEE, K. S. & LEE, Y. C. 2003. Involvement of PTEN in airway hyperresponsiveness and inflammation in bronchial asthma. J Clin Invest, 111, 1083-92.

LALKO, J. F., KIMBER, I., DEARMAN, R. J., API, A. M. & GERBERICK, G. F. 2013. The selective peptide reactivity of chemical respiratory allergens under competitive and non-competitive conditions. J Immunotoxicol, 10, 292-301.

LALKO, J. F., KIMBER, I., DEARMAN, R. J., GERBERICK, G. F., SARLO, K. & API, A. M. 2011. Chemical reactivity measurements: potential for characterization of respiratory chemical allergens. Toxicol In Vitro, 25, 433-45.

LAUENSTEIN, L., SWITALLA, S., PRENZLER, F., SEEHASE, S., PFENNIG, O., FÖRSTER, C., FIEGUTH, H., BRAUN, A. & SEWALD, K. 2014. Assessment of immunotoxicity induced by chemicals in human precision-cut lung slices (PCLS). Toxicol In Vitro, 28, 588-99.

NATSCH, A., RYAN, C. A., FOERTSCH, L., EMTER, R., JAWORSKA, J., GERBERICK, F. & KERN, P. 2013. A dataset on 145 chemicals tested in alternative assays for skin sensitization undergoing prevalidation. J Appl Toxicol, 33, 1337-52.

REMY, S., VERSTRAELEN, S., VAN DEN HEUVEL, R., NELISSEN, I., LAMBRECHTS, N., HOOYBERGHS, J. & SCHOETERS, G. 2014. Gene expressions changes in bronchial epithelial cells: markers for respiratory sensitizers and exploration of the NRF2 pathway. Toxicol In Vitro, 28, 209-17.

SEED, M. & AGIUS, R. 2010. Further validation of computer-based prediction of chemical asthma hazard. Occup Med (Lond), 60, 115-20.

SEED, M. J. & AGIUS, R. M. 2017. Progress with Structure-Activity Relationship modelling of occupational chemical respiratory sensitizers. Curr Opin Allergy Clin Immunol, 17, 64-71.

VERSTRAELEN, S., NELISSEN, I., HOOYBERGHS, J., WITTERS, H., SCHOETERS, G., VAN CAUWENBERGE, P. & VAN DEN HEUVEL, R. 2009. Gene profiles of a human alveolar epithelial cell line after in vitro exposure to respiratory (non-)sensitizing chemicals: identification of discriminating genetic markers and pathway analysis. Toxicol Lett, 185, 16-22.