Development Of Adverse Outcome Pathways Research Articles

Methodology for developing data-rich Key Event Relationships for Adverse Outcome Pathways exemplified by linking decreased androgen receptor activity with decreased anogenital distance

The Adverse Outcome Pathway (AOP) framework has gained widespread acceptance in toxicological disciplines as a tool for aiding chemical hazard assessment. Despite increased activity in AOP development, progress towards a high volume of fully endorsed AOPs has been slow, partly due to the challenging task of constructing complete AOPs according to the AOP Developer’s Handbook. To facilitate greater uptake of new knowledge units onto the open-source AOP-wiki platform, a pragmatic approach was recently proposed. This approach involves considering Key Event Relationships (KERs) for individual development through systematic approaches, as they represent essential units of knowledge from which causality can be inferred; from low complexity test data to adverse outcomes in intact organisms. However, more broadly adopted harmonized methodologies for KER development would be desirable. Using the AOP Developer’s Handbook as a guide, a KER linking 'decreased androgen receptor (AR) activity' with 'reduced anogenital distance (AGD)' was developed to demonstrate a methodology applicable for future developments of KERs requiring systematic literature retrieval approaches.

Outsourcing preparatory work based on a systematic literature review for the development of adverse outcome pathways (AOPs) relevant for the capacity of proteins to trigger celiac disease

Outsourcing preparatory work based on a systematic literature review for the development of adverse outcome pathways (AOPs) relevant for the capacity of proteins to trigger celiac disease

Identification of neurotoxicology (NT)/developmental neurotoxicology (DNT) adverse outcome pathways and key event linkages with in vitro DNT screening assays

There is a need to assess compounds reliably and quickly for neurotoxicity (NT) and developmental neurotoxicity (DNT). Adverse outcome pathways (AOPs) enable the mapping of molecular events to an apical endpoint in a chemical agnostic manner and have begun to be applied in NT and DNT testing frameworks. We assessed the status of NT/DNT AOPs in the AOP-Wiki (ca. 2/1/23; https://aopwiki.org/), to characterize the state of AOP development, identify strengths and knowledge gaps, elucidate areas for improvement, and describe areas for future focus. AOPs in the Wiki database were assessed for inclusion of NT/DNT molecular events and endpoints, AOP development and endorsement, as well as the linkages of key neurodevelopmental processes with in vitro new approach methods (NAMs). This review found that 41 AOPs have been proposed detailing NT/DNT, of which eight were endorsed by working parties in OECD. Further, this review determined that learning and memory is included as an adverse outcome in eight NT/DNT AOPS, often without distinction regarding the varying forms of learning and memory, regional specification, temporal dynamics, or acquisition mechanisms involved. There is also an overlap with key events (KEs) and in vitro NAMs, which synaptogenesis appeared as a common process. Overall, progress on NT/DNT AOPs could be expanded, adding in modes of action that are missing, improvement in defining apical endpoints, as well as utilizing NAMs further to develop AOPs and identify gaps in current knowledge.

P01-07: EFSA activities in the development of Adverse Outcome Pathways for the identification of substances having endocrine disruption properties

P01-07: EFSA activities in the development of Adverse Outcome Pathways for the identification of substances having endocrine disruption properties

Unveiling potentially convergent key events related to adverse outcome pathways induced by silver nanoparticles via cross-species omics-scale analysis

The adverse effects of silver nanoparticles (AgNPs) have been studied in various models. However, there has been discordance between molecular responses across the literature, attributed to methodological biases and the physicochemical variability of AgNPs. In this study, a gene pathway meta-analysis was conducted to identify convergent and divergent key events (KEs) associated with AgNPs and explore common patterns of these KEs across species. We performed a cross-species analysis of transcriptomic data from multiple studies involving various AgNPs exposure. Pathway enrichment analysis revealed a set of pathways linked to oxidative stress, apoptosis, and metabolite and lipid metabolism, which are considered potentially conserved KEs across species. Subsequently, experiments confirmed that oxidative stress responses could be early KEs in both Caenorhabditis elegans and HepG2 cells. Moreover, AgNPs preferentially impaired the mitochondria, as evidenced by mitochondrial fragmentation and dysfunction. Furthermore, disruption of amino acids, nucleotides, sulfur compounds, glycerolipids, and glycerophospholipids metabolism were in good agreement with gene pathway shreds of evidence. Our findings imply that, although there may be organism-specific responses, potentially conserved events could exist regardless of species and physicochemical factors. These results provide valuable insights into the development of adverse outcome pathways of AgNPs across species and the regulatory toxicity of AgNPs.

An integrative systems biology strategy to support the development of adverse outcome pathways (AOPs): a case study on radiation-induced microcephaly.

Adverse Outcome Pathways (AOPs) are useful tools for assessing the potential risks associated with exposure to various stressors, including chemicals and environmental contaminants. They provide a framework for understanding the causal relationships between different biological events that can lead to adverse outcomes (AO). However, developing an AOP is a challenging task, particularly in identifying the molecular initiating events (MIEs) and key events (KEs) that constitute it. Here, we propose a systems biology strategy that can assist in the development of AOPs by screening publicly available databases, literature with the text mining tool AOP-helpFinder, and pathway/network analyses. This approach is straightforward to use, requiring only the name of the stressor and adverse outcome to be studied. From this, it quickly identifies potential KEs and literature providing mechanistic information on the links between the KEs. The proposed approach was applied to the recently developed AOP 441 on radiation-induced microcephaly, resulting in the confirmation of the KEs that were already present and identification of new relevant KEs, thereby validating the strategy. In conclusion, our systems biology approach represents a valuable tool to simplify the development and enrichment of Adverse Outcome Pathways (AOPs), thus supporting alternative methods in toxicology.

Putative adverse outcome pathway development based on physiological responses of female fathead minnows to model estrogen versus androgen receptor agonists

Several adverse outcome pathways (AOPs) have linked molecular initiating events like aromatase inhibition, androgen receptor (AR) agonism, and estrogen receptor (ER) antagonism to reproductive impairment in adult fish. Estrogen receptor agonists can also cause adverse reproductive effects, however, the early key events (KEs) in an AOP leading to this are mostly unknown. The primary aim of this study was to develop hypotheses regarding the potential mechanisms through which exposure to ER agonists might lead to reproductive impairment in female fish. Mature fathead minnows were exposed to 1 or 10 ng 17α-ethynylestradiol (EE2)/L or 10 or 100 µg bisphenol A (BPA)/L for 14 d. The response to EE2 and BPA was contrasted with the effects of 500 ng/L of 17β-trenbolone (TRB), an AR agonist, as well as TRB combined with the low and high concentrations of EE2 or BPA tested individually. Exposure to 10 ng EE2/L, 100 µg BPA/L, TRB, or the various mixtures with TRB caused significant decreases in plasma concentrations of 17β-estradiol. Exposure to TRB alone caused a significant reduction in plasma vitellogenin (VTG), but VTG was unaffected or even increased in females exposed to EE2 or BPA alone or, in most cases, in mixtures with TRB. Over the course of the 14-d exposure, the only treatments that clearly did not affect egg production were 1 ng EE2/L and 10 µg BPA/L. Based on these results and knowledge of hypothalamic–pituitary–gonadal axis function, we hypothesize an AOP whereby decreased production of maturation-inducing steroid leading to impaired oocyte maturation and ovulation, possibly due to negative feedback or direct inhibitory effects of membrane ER activation, could be responsible for causing adverse reproductive impacts in female fish exposed to ER agonists.

89 Lung Single Cell Transcriptomics to Guide the Development of AOP Anchored Cell-Based Assays in Response to Nanoparticle Exposure

Abstract Inhaled nanoparticles (NP) can cause acute and chronic inflammation. Here we seek to identify NP-specific cellular perturbation pathways and the underlying key cell types to inform adverse outcome pathway (AOP) anchored in vitro studies, by single cell transcriptomics. We exposed mice intratracheally to carbon black (CNP), tangled double-walled (DWCNT) and rigid, multi-walled carbon nanotubes (MWCNT). All lungs underwent single-cell RNA sequencing (scRNA-seq), histology and BAL analysis. At the chosen doses all NPs caused comparable airspace neutrophilia at 12h, which increased until d6 for the CNTs but remained elevated until d28 only for MWCNT, indicating the key event (KE) acute and chronic inflammation. Comparing scRNA-seq and BAL cytokine levels at 12h, differentiated the fate of inflammation NP specifically. In agreement with airspace neutrophilia, CNP uniquely triggered GM-CSF and CXCL1 release, with Csf2 and Cxcl1 mRNAs being expressed mainly in alveolar epithelial cells. DWCNT caused CCL2, -3 and -4 release involving interstitial macrophages and monocytes, associated with acute high lung monocyte and at d6 high BAL macrophage numbers. MWCNT in contrast caused ample BAL cytokine increases involving different cell types and specifically caused a Th2 cytokine response (CCL11, IL10), a crucial KE of fibrosis AOPs. MWCNT triggered cell communication analysis uncovered NP-specific networks including epithelial cells, fibroblasts, macrophages, dendritic cells and endothelial cells. Our study uncovers early NP-specific cell perturbations and identified first specific cellular response pattern to guide AOP predictive cell-based testing strategies. Further analysis of the cellular dynamics shall enhance our understanding of NP specific cell circuits and related pathologies.

Triazophos-induced Respiratory and Behavioral Effects and Development of Adverse Outcome Pathway (AOP) for short-term Exposed Freshwater Snail, Bellamya Bengalensis.

The physiological effects of triazophos were examined using respiratory and behavioral endpoints in Bellamya bengalensis under a 96-hour acute exposure regime. Physiological manifestation of respiratory stress was measured using the rate of oxygen consumption while behavioral toxicity was measured using crawling reflexes, touch response, and mucus production. The threshold effect values for LOEC (Lowest Observed Effect Concentration), NOEC (No Observed Effect Concentration), and MATC (Maximum Acceptable Toxicant Concentration) at 96h were 0.40, 0.60, and 0.075mg/l, respectively. Definitive 96h acute exposures for both respiratory and behavioral endpoints tests were determined using a control group and concentrations ranging from 0.40 to 1.60mg/l monitored for 24, 48, 72, and 96h. Test organisms irrespective of exposure concentration demonstrated an initial rise in oxygen consumption rate after 24h, followed by a progressive decrease in toxicant concentration and exposure period. The in silico structural analysis presents triazophos as having an electrophilic toxic structure similar to choline esterase inhibitors, and also capable of inducing oxidative stress. The AOP highlighted neurotoxicity and oxidative stress as plausible pathways of triazophos toxicity in mollusk species.

Identification of molecular initiating events (MIE) using chemical database analysis and nuclear receptor activity assays for screening potential inhalation toxicants

An adverse outcome pathway (AOP) framework can facilitate the use of alternative assays in chemical regulations by providing scientific evidence. Previously, an AOP, peroxisome proliferative-activating receptor gamma (PPARγ) antagonism that leads to pulmonary fibrosis, was developed. Based on a literature search, PPARγ inactivation has been proposed as a molecular initiating event (MIE). In addition, a list of candidate chemicals that could be used in the experimental validation was proposed using toxicity database and deep learning models. In this study, the screening of environmental chemicals for MIE was conducted using in silico and in vitro tests to maximize the applicability of this AOP for screening inhalation toxicants. Initially, potential inhalation exposure chemicals that are active in three or more key events were selected, and in silico molecular docking was performed. Among the chemicals with low binding energy to PPARγ, nine chemicals were selected for validation of the AOP using in vitro PPARγ activity assay. As a result, rotenone, triorthocresyl phosphate, and castor oil were proposed as PPARγ antagonists and stressor chemicals of the AOP. Overall, the proposed tiered approach of the database-in silico-in vitro can help identify the regulatory applicability and assist in the development and experimental validation of AOP.

Prediction of Molecular Initiating Events for Adverse Outcome Pathways Using High-Throughput Identification of Chemical Targets.

The impact of exposure to multiple chemicals raises concerns for human and environmental health. The adverse outcome pathway method offers a framework to support mechanism-based assessment in environmental health starting by describing which mechanisms are triggered upon interaction with different stressors. The identification of the molecular initiating event and the molecular interaction between a chemical and a protein target is still a challenge for the development of adverse outcome pathways. The cellular response to chemical exposure studied with omics could not directly identify the protein targets. However, recent mass spectrometry-based methods are offering a proteome-wide identification of protein targets interacting with s but unrevealing a molecular initiating event from a set of targets is still dependent on available knowledge. Here, we directly coupled the target identification findings from the proteome integral solubility alteration assay with an analytical hierarchy process for the prediction of a prioritized molecular initiating event. We demonstrate the applicability of this combination of methodologies with a test compound (TCDD), and it could be further studied and integrated into AOPs. From the eight protein targets identified by the proteome integral solubility alteration assay after analyzing 2824 human hepatic proteins, the analytical hierarchy process can select the most suitable protein for an AOP. Our combined method solves the missing links between high-throughput target identification and prediction of the molecular initiating event. We anticipate its utility to decipher new molecular initiating events and support more sustainable methodologies to gain time and resources in chemical assessment.

Multiomics Point of Departure (moPOD) Modeling Supports an Adverse Outcome Pathway Network for Ionizing Radiation.

While adverse biological effects of acute high-dose ionizing radiation have been extensively investigated, knowledge on chronic low-dose effects is scarce. The aims of the present study were to identify hazards of low-dose ionizing radiation to Daphnia magna using multiomics dose-response modeling and to demonstrate the use of omics data to support an adverse outcome pathway (AOP) network development for ionizing radiation. Neonatal D. magna were exposed to γ radiation for 8 days. Transcriptomic analysis was performed after 4 and 8 days of exposure, whereas metabolomics and confirmative bioassays to support the omics analyses were conducted after 8 days of exposure. Benchmark doses (BMDs, 10% benchmark response) as points of departure (PODs) were estimated for both dose-responsive genes/metabolites and the enriched KEGG pathways. Relevant pathways derived using the BMD modeling and additional functional end points measured by the bioassays were overlaid with a previously published AOP network. The results showed that several molecular pathways were highly relevant to the known modes of action of γ radiation, including oxidative stress, DNA damage, mitochondrial dysfunction, protein degradation, and apoptosis. The functional assays showed increased oxidative stress and decreased mitochondrial membrane potential and ATP pool. Ranking of PODs at the pathway and functional levels showed that oxidative damage related functions had relatively low PODs, followed by DNA damage, energy metabolism, and apoptosis. These were supportive of causal events in the proposed AOP network. This approach yielded promising results and can potentially provide additional empirical evidence to support further AOP development for ionizing radiation.

Development of Adverse Outcome Pathways relevant for the identification of substances having endocrine disruptors properties

This report is the outcome of an EFSA procurement (NP/EFSA/PREV/2020/01) which aims at contributing to the development of adverse outcome pathways (AOPs) to be integrated in a network addressing uterine adenocarcinoma in mammals. The outcome is intended to support the identification of substances with endocrine disruptor mode of action. For this specific purpose, an evidence-based approach methodology was adopted. Available evidence was systematically mapped from the literature to identify Molecular Initiating Events (MIEs) and Key Events (KEs) linked to adverse outcome (AO) “uterine endometrioid adenocarcinoma” independently from prototypical stressors, by means of: 1. a priori defined search strategies initially addressing the AO and biologically plausible MIEs, 2. application of machine learning technique (Topic modelling) that automatically analyzes text data to identify biologically plausible KERs, 3. systematic literature review and critical appraisal of prioritized evidence, taking into account human, in vivo and in vitro studies. Estradiol and tamoxifen, two recognized human risk factors for endometrioid adenocarcinoma, were used as tool chemical compounds to empirically support the response and temporal concordance of the identified key event relationships (KERs). All evidence has been then integrated by means of the AOP conceptual network. An evidence-based AOP starting from activation of uterine estrogen receptor-alfa leading to endometrial adenocarcinoma via epigenetic modulation was postulated.

Development of adverse outcome pathways relevant for the identification of substances having endocrine disruption properties Uterine adenocarcinoma as adverse outcome.

Development of adverse outcome pathways (AOPs) for uterine adenocarcinoma can provide a practical tool to implement the EFSA-ECHA Guidance (2018) for the identification of endocrine disruptors in the context of Regulations (EU) No 528/2012 and (EC) No 1107/2009. AOPs can give indications about the strength of the relationship between an adverse outcome (intended as a human health outcome) and chemicals (pesticides but not only) affecting the pathways. In this scientific opinion, the PPR Panelexplored the development of AOPs for uterine adenocarcinoma. An evidence-based approach methodology was applied, and literature reviews were produced using a structured framework assuring transparency, objectivity, and comprehensiveness. Several AOPs were developed; these converged to a common critical node, that is increased estradiol availability in the uterus followed by estrogen receptor activation in the endometrium; therefore, a putative AOP network was considered. An uncertainty analysis and a probabilistic quantification of the weight of evidence have been carried out via expert knowledge elicitation for each set of MIEs/KEs/KERs included in individual AOPs. The collected data on the AOP network were evaluated qualitatively, whereas a quantitative uncertainty analysis for weight of the AOP network certainty has not been performed. Recommendations are provided, including exploring further the uncertainties identified in the AOPs and putative AOP network; further methodological developments for quantifying the certainty of the KERs and of the overall AOPs and AOP network; and investigating of NAMs applications in the context of some of the MIEs/KEs currently part of the putative AOP network developed.

Combining time-resolved transcriptomics and proteomics data for Adverse Outcome Pathway refinement in ecotoxicology

Conventional Environmental Risk Assessment (ERA) of pesticide pollution is based on soil concentrations and apical endpoints, such as the reproduction of test organisms, but has traditionally disregarded information along the organismal response cascade leading to an adverse outcome. The Adverse Outcome Pathway (AOP) framework includes response information at any level of biological organization, providing opportunities to use intermediate responses as a predictive read-out for adverse outcomes instead. Transcriptomic and proteomic data can provide thousands of data points on the response to toxic exposure. Combining multiple omics data types is necessary for a comprehensive overview of the response cascade and, therefore, AOP development. However, it is unclear if transcript and protein responses are synchronized in time or time lagged. To understand if analysis of multi-omics data obtained at the same timepoint reveal one synchronized response cascade, we studied time-resolved shifts in gene transcript and protein abundance in the springtail Folsomia candida, a soil ecotoxicological model, after exposure to the neonicotinoid insecticide imidacloprid. We analyzed transcriptome and proteome data every 12 h up to 72 h after onset of exposure. The most pronounced shift in both transcript and protein abundances was observed after 48 h exposure. Moreover, cross-correlation analyses indicate that most genes displayed the highest correlation between transcript and protein abundances without a time-lag. This demonstrates that a combined analysis of transcriptomic and proteomic data from the same time-point can be used for AOP improvement. This data will promote the development of biomarkers for the presence of neonicotinoid insecticides or chemicals with a similar mechanism of action in soils.

Beyond chemicals: Opportunities and challenges of integrating non-chemical stressors in adverse outcome pathways.

The adverse outcome pathways (AOPs) were developed to accelerate evidence-based chemical risk assessment by leveraging data from new approach methodologies. Thanks to their stressor-agnostic approach, AOPs were seen as instrumental in other fields. Here, we present AOPs that report non-chemical stressors along with the challenges encountered for their development. Challenges regarding AOPs linked to nanomaterials include non-specific molecular initiating events, limited understanding of nanomaterial biodistribution, and needs for adaptations of the in silico modeling and testing systems. Development of AOPs for radiation face challenges in how to incorporate ionizing events type, dose rate, energy deposition, and how to account for targeting multiple macromolecules. AOPs for COVID-19 required the inclusion of SARS-CoV-2-specific replicative steps to capture the essential events driving the disease. Developing AOPs to evaluate efficacy and toxicity of cell therapies necessitates addressing the cellular nature and the therapeutic function of the stressor. Finally, addressing toxicity of emerging biological stressors like microbial pesticides can learn from COVID-19 AOPs. We further discuss that the adaptations needed to expand AOP applicability beyond chemicals are mainly at the molecular and cellular levels while downstream key events at tissue or organ level, such as inflammation, are shared by many AOPs initiated by various stressors. In conclusion, although it is challenging to integrate non-chemical stressors within AOPs, this expands opportunities to account for real-world scenarios, to identify vulnerable individuals, and to bridge knowledge on mechanisms of adversity.

Radiation adverse outcome pathways (AOPs) are on the horizon: advancing radiation protection through an international Horizon-Style exercise

Purpose The Adverse Outcome Pathway (AOP) framework, a systematic tool that can link available mechanistic data with phenotypic outcomes of relevance to regulatory decision-making, is being explored in areas related to radiation risk assessment. To examine the challenges including the use of AOPs to support the radiation protection community, an international horizon-style exercise was initiated through the Organisation for Economic Co-operation and Development Nuclear Energy Agency High-Level Group on Low Dose Research Radiation/Chemical AOP Joint Topical Group. The objective of the HSE was to facilitate the collection of ideas from a range of experts, to short-list a set of priority research questions that could, if answered, improve the description of the radiation dose-response relationship for low dose/dose-rate exposures, as well as reduce uncertainties in estimating the risk of developing adverse health outcomes following such exposures. Materials and methods The HSE was guided by an international steering committee of radiation risk experts. In the first phase, research questions were solicited on areas that can be supported by the AOP framework, or challenges on the use of AOPs in radiation risk assessment. In the second phase, questions received were refined and sorted by the SC using a best-worst scaling method. During a virtual 3-day workshop, the list of questions was further narrowed. In the third phase, an international survey of the broader radiation protection community led to an orderly ranking of the top questions. Results Of the 271 questions solicited, 254 were accepted and categorized into 9 themes. These were further refined to the top 25 prioritized questions. Among these, the higher ranked questions will be considered as ‘important’ to drive future initiatives in the low dose radiation protection community. These included questions on the ability of AOPs to delineate responses across different levels of biological organization, and how AOPs could be applied to address research questions on radiation quality, doses or dose-rates, exposure time patterns and deliveries, and uncertainties in low dose/dose-rate effects. A better understanding of these concepts is required to support the use of the AOP framework in radiation risk assessment. Conclusion Through dissemination of these results and considerations on next steps, the JTG will address select priority questions to advance the development and use of AOPs in the radiation protection community. The major themes observed will be discussed in the context of their relevance to areas of research that support the system of radiation protection.

Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example

Purpose Health protection agencies require scientific information for evidence-based decision-making and guideline development. However, vetting and collating large quantities of published research to identify relevant high-quality studies is a challenge. One approach to address this issue is the use of adverse outcome pathways (AOPs) that provide a framework to assemble toxicological knowledge into causally linked chains of key events (KEs) across levels of biological organization to culminate in an adverse health outcome of significance to regulatory decision-making. Traditionally, AOPs have been constructed using a narrative review approach where the collection of evidence that supports each pathway is based on prior knowledge of influential studies that can also be supplemented by individually selecting and reviewing relevant references. Objectives We aimed to create a protocol for AOP weight of evidence gathering that harnesses elements of both scoping review methods and artificial intelligence (AI) tools to increase transparency while reducing bias and workload of human screeners. Methods To develop this protocol, an existing space-health AOP in the workplan of the Organisation for Economic Co-operation and Development (OECD) AOP Programme was used as a case example. To balance the benefits of both scoping review tools and narrative approaches, a study protocol outlining a screening and search strategy was developed, and three reference collection workflows were tested to identify the most efficient method to inform weight of evidence. The workflows differed in their literature search strategies, and combinations of software tools used. Results Across the three tested workflows, over 59 literature searches were completed, retrieving over 34,000 references of which over 3300 were human reviewed. The most effective of the three methods used a search strategy with searches across each component of the AOP network, SWIFT Review as a pre-filtering software, and DistillerSR to create structured screening and data extraction forms. This methodology effectively retrieved relevant studies while balancing efficiency in data retrieval without compromising transparency, leading to a well-synthesized evidence base to support the AOP. Conclusions The workflow is still exploratory in the context of AOP development, and we anticipate adaptations to the protocol with further experience. To further the systematicity, future iterations of the workflow could include structured quality assessment and risk of bias analysis. Overall, the workflow provides a transparent and documented approach to support AOP development, which in turn will support the need for rigorous methods to identify relevant scientific evidence while being practical to allow uptake by the broader community.

Adverse outcome pathways and linkages to transcriptomic effects relevant to ionizing radiation injury

Background In the past three decades, a large body of data on the effects of exposure to ionizing radiation and the ensuing changes in gene expression has been generated. These data have allowed for an understanding of molecular-level events and shown a level of consistency in response despite the vast formats and experimental procedures being used across institutions. However, clarity on how this information may inform strategies for health risk assessment needs to be explored. An approach to bridge this gap is the adverse outcome pathway (AOP) framework. AOPs represent an illustrative framework characterizing a stressor associated with a sequential set of causally linked key events (KEs) at different levels of biological organization, beginning with a molecular initiating event (MIE) and culminating in an adverse outcome (AO). Here, we demonstrate the interpretation of transcriptomic datasets in the context of the AOP framework within the field of ionizing radiation by using a lung cancer AOP (AOP 272: https://www.aopwiki.org/aops/272) as a case example. Methods Through the mining of the literature, radiation exposure-related transcriptomic studies in line with AOP 272 related to lung cancer, DNA damage response, and repair were identified. The differentially expressed genes within relevant studies were collated and subjected to the pathway and network analysis using Reactome and GeneMANIA platforms. Identified pathways were filtered (p < .001, ≥3 genes) and categorized based on relevance to KEs in the AOP. Gene connectivities were identified and further grouped by gene expression-informed associated events (AEs). Relevant quantitative dose-response data were used to inform the directionality in the expression of the genes in the network across AEs. Results Reactome analyses identified 7 high-level biological processes with multiple pathways and associated genes that mapped to potential KEs in AOP 272. The gene connectivities were further represented as a network of AEs with associated expression profiles that highlighted patterns of gene expression levels. Conclusions This study demonstrates the application of transcriptomics data in AOP development and provides information on potential data gaps. Although the approach is new and anticipated to evolve, it shows promise for improving the understanding of underlying mechanisms of disease progression with a long-term vision to be predictive of adverse outcomes.

Adverse outcome pathways (AOPs) for radiation-induced reproductive effects in environmental species: state of science and identification of a consensus AOP network

Background Reproductive effects of ionizing radiation in organisms have been observed under laboratory and field conditions. Such assessments often rely on associations between exposure and effects, and thus lacking a detailed mechanistic understanding of causality between effects occurring at different levels of biological organization. The Adverse Outcome Pathway (AOP), a conceptual knowledge framework to capture, organize, evaluate and visualize the scientific knowledge of relevant toxicological effects, has the potential to evaluate the causal relationships between molecular, cellular, individual, and population effects. This paper presents the first development of a set of consensus AOPs for reproductive effects of ionizing radiation in wildlife. This work was performed by a group of experts formed during a workshop organized jointly by the Multidisciplinary European Low Dose Initiative (MELODI) and the European Radioecology Alliance (ALLIANCE) associations to present the AOP approach and tools. The work presents a series of taxon-specific case studies that were used to identify relevant empirical evidence, identify common AOP components and propose a set of consensus AOPs that could be organized into an AOP network with broader taxonomic applicability. Conclusion Expert consultation led to the identification of key biological events and description of causal linkages between ionizing radiation, reproductive impairment and reduction in population fitness. The study characterized the knowledge domain of taxon-specific AOPs, identified knowledge gaps pertinent to reproductive-relevant AOP development and reflected on how AOPs could assist applications in radiation (radioecological) research, environmental health assessment, and radiological protection. Future advancement and consolidation of the AOPs is planned to include structured weight of evidence considerations, formalized review and critical assessment of the empirical evidence prior to formal submission and review by the OECD sponsored AOP development program.