Table 1.
We defined characteristics for optimal biomarkers based on the Bradford Hill criteria. The characteristics were then implemented into the prioritization and selection protocol, and further to the evaluation of the prioritized genes
| Bradford Hill | Our characteristic | Method/Assessment |
|---|---|---|
| Consistency (reproducibility) | Reproducibility | Selection considers evidence from previous profibrotic exposures |
| Strength (effect size) | Amplitude | Significant alteration of the expression as compared to control |
| Experiment | Measurable | Transcriptional biomarkers measurable by qPCR; selected genes need to be expressed in the model |
| Biological gradient (dose‐response relationship) | Dose‐responsive | Benchmark dose modeling to evaluate dose‐response |
| Coherence | In vitro to in vivo extrapolation | Experimental evidence from in vitro and in vivoa) |
| Analogy | Predictive (of the outcome of interest) | Selection based on the KEs preceding the AO of interest |
| Specificity | Specificity | Gene ranking based on the specificity score |
| Plausibility | (Biological) plausibility | The AOP framework provides a plausible context; supporting evidence; selection of the organism |
| Temporality | Temporality | Transcriptional alteration follows the exposure; selection of the model organismb) |
| – | GLP‐method | RT‐qPCR |
| – | Influence | Centrality measures from human protein‐protein interaction and gene regulatory networks |
The biomarkers selected here are targeted for the development of non‐animal assays for toxicological assessment. Hence the coherence to in vivo set ups is not evaluated experimentally. However, in vivo data was used for the selection of the markers to provide context of the systemic response
Temporality in the Bradford Hill criteria refers to a clear distinction of the exposure happening prior to the outcome. Here, we considered temporality by observing transcriptional changes post exposure as well as in the selection of the model organism. Macrophages have a crucial role in the initiation of the profibrotic response preceding the outcome, fibrosis.