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Table 2 Key events (KE) contained in AOP 173 (https://aopwiki.org/aops/173). Markers, cell types and assays were identified in Vietti et al., 2016 and Nymark et al., 2018. WP numbers refer to Wikipathways (https://www.wikipathways.org/index.php/WikiPathways). Relevance of the biomarkers for predicting lung fibrosis is described as A (association between in vitro and in vivo data for the biomarker), B (biomarker involved in the AO as demonstrated with deficient or transgenic mice, inhibitors, etc), C (biomarker strongly associated with the AO), D (biomarker identified by data mining). ELISA: enzyme-linked immunosorbent assays; EPR: electron paramagnetic resonance; GO: Gene Ontologies; qRT-PCR: quantitative reverse transcription-polymerase reaction; WB: western blot

From: Adverse outcome pathways as a tool for the design of testing strategies to support the safety assessment of emerging advanced materials at the nanoscale

KE #KEBiomarkersCell typeAssayRelevance
1495Interaction with the resident cell membrane componentsToll-like receptor signaling WP75 (CXCL8, CCL3, CCL4, CCL5)Epithelial cellsTranscriptomics or individual assays (qRT-PCR)D
DAMPS/alarmins (IL-1a)MacrophagesELISA, qRT-PCRC
1496Secretion of proinflammatory and profibrotic mediatorsROSMacrophages, fibroblastsEPR (acellular), HO-1 (cellular, ELISA, RT-PCR)C
p38 MAPKFibroblastsWBC
NF-KBMacrophagesWBC
MAP kinaseEpithelial cellsWBC
IL-1b (+ NADPH oxidase and inflammasome)Macrophages, epithelial cellsELISA, WB (± NADPH oxidase or inflammasome inhibitors)A, B, C
TNF-aMacrophagesELISA, WB (qRT-PCR)C
IL-18Epithelial cellsELISA, WB (qRT-PCR)C
IL-8Epithelial cellsELISA, WB (qRT-PCR)C
TGF-bMacrophages, fibroblasts, epithelial cellsELISA, WB (qRT-PCR)B, C
PDGFMacrophages, fibroblasts, epithelial cellsELISA, WB (qRT-PCR)C
Cytokine and inflammatory response WP530 (PDGFA, CXCL2, CSF3, CSF2, IL12B, IL13, IL4, IL5, IL6)Epithelial cellsTranscriptomicsD
Chemokine signaling WP3929 (CCL2, CCL11, CCR2, CCR3)Epithelial cellsTranscriptomicsD
1497Recruitment of inflammatory cells    
1498Loss of alveolar capillary membrane integrityTransepithelial/transendothelial electrical resistance (TEER)Endothelial cells, epithelial cellsOhmic resistance or impedanceC
ROSMacrophages, fibroblastsEPR (acellular), HO-1 (cellular, ELISA, qRT-PCR)C
1499Activation of T (T) helper (h) type 2 cellsChondrocyte differentiation WP474 (CTGF, TGFA, GREM1, ATP11A)Epithelial cellsTranscriptomicsD
Matrix metalloproteinases WP129 (MMP9, MMP2, TIMP1)Epithelial cellsTranscriptomicsD
TGFB signaling WP560 (SKIL, SPP1)Epithelial cellsTranscriptomicsD
Differentiation pathway WP2848 (EFG, IGF1, HGF, FGF1, FGF2, FGF7)Epithelial cellsTranscriptomicsD
Cytokine and inflammatory response WP530 (PDGFA, CXCL2, CSF3, CSF2, IL12B, IL13, IL4, IL5, IL6)Epithelial cellsTranscriptomicsD
Chemokine signaling WP3929 (CCL2, CCL11, CCR2, CCR3)Epithelial cellsTranscriptomicsD
Leukocyte/Myeloid cell differentiation GO: 0045637/GO: 1902105 (CALCA, CEBPB)Epithelial cellsTranscriptomicsD
TGF-bMacrophages, fibroblasts, epithelial cellsELISA, WB (qRT-PCR)B, C, D
PDGFMacrophages, fibroblasts, epithelial cellsELISA, WB (qRT-PCR)C, D
1500Fibroblast proliferation and myofibroblast differentiationSmadFibroblasts, epithelial cellsWBC
ERK1/2FibroblastsWBA
fibroblast proliferationFibroblastscell count, cell viability assaysA
fibroblast differentiation (a-SMA)FibroblastsqRT-PCR, WBC
epithelial-mesenchymal transition, EMT (ZO-1, SP-C, E-Cad, fibronectin, FSP-1, a-SMA, vimentin)Epithelial cellsqRT-PCR, WBC
MAPK signaling WP382Epithelial cellsTranscriptomicsD
p38 MAPK WP400Epithelial cellsTranscriptomicsD
TGFB signaling WP560 (SKIL, SPP1)Epithelial cellsTranscriptomicsD
TGF-bMacrophages, fibroblasts, epithelial cellsELISA, WB (qRT-PCR)B, C, D
PDGFMacrophages, fibroblasts, epithelial cellsELISA, WB (qRT-PCR)C, D
Chondrocyte differentiation WP474 (CTGF, TGFA, GREM1, ATP11A)Epithelial cellsTranscriptomicsD
Differentiation pathway WP2848 (EFG, IGF1, HGF, FGF1, FGF2, FGF7)Epithelial cellsTranscriptomicsD
1501Extracellular matrix depositionCollagen production (Collagen I and III or soluble collagen)FibroblastsqRT-PCR, WB, Sircol assayA
1458Pulmonary fibrosis