Reactive oxygen species leading to growth inhibition via lipid peroxidation and cell death

You Song, Li Xie, Knut Erik Tollefsen

Norwegian Institute for Water Research (NIVA), Sognsveien 72, 0855, Oslo, Norway

This adverse outcome pathway (AOP 331) describes a linear route by which increased reactive oxygen species (ROS) can lead to decreased organismal growth through lipid peroxidation-mediated mitochondrial bioenergetic impairment and increased cell injury/death. In this AOP, increased ROS is treated operationally as the molecular initiating event because it represents the earliest common measurable redox perturbation shared by many chemical and non-chemical stressors within the broader ROS-growth AOP network. Increased ROS leads to oxidative stress, which promotes lipid peroxidation. Oxidative damage to membrane lipids can impair mitochondrial membrane integrity and coupling of oxidative phosphorylation (OXPHOS). Decreased OXPHOS coupling reduces ATP production, and insufficient ATP availability can compromise membrane homeostasis, ion transport, biosynthesis, stress-response capacity, and execution of regulated cell death pathways, ultimately resulting in increased cell injury/death. Increased loss of viable cells, particularly in developing, growing, or regenerating tissues and organisms, can contribute to decreased growth.

AOP 331 reuses and connects established AOP-Wiki components from several AOP contexts. The upstream ROS and oxidative stress segment is associated with AOP 478, in which deposition of energy leads to oxidative stress through increased free radical generation (AOP-Wiki, 2026a). The lipid peroxidation and mitochondrial bioenergetic segment is connected to the oxidative stress and mitochondrial impairment logic represented in the broader ROS-growth AOP network, while the KER from decreased coupling of OXPHOS to decreased ATP pool is directly associated with AOP 263, an OECD-published AOP that causally links uncoupling of OXPHOS to growth inhibition through ATP depletion and decreased cell proliferation (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021). AOP 331 differs from AOP 326 by routing ATP depletion through increased cell injury/death rather than decreased cell proliferation. This terminal cellular injury module is supported by reuse of the broadly shared AOP-Wiki KE 'Increase, Cell injury/death' and by its occurrence in several AOPs, including AOPs 12, 13, 17, 38, and 48, where cell injury/death is used as an intermediate or downstream KE in neurotoxicity, oxidative stress, fibrosis, and excitotoxicity contexts (AOP-Wiki, 2026c-g). The AOP is relevant to environmental and human health contexts because ROS production, lipid peroxidation, mitochondrial ATP production, cell viability, and growth are conserved biological processes. It can support mechanistic interpretation of oxidative stress-mediated growth impairment, assay selection, chemical prioritization, integrated approaches to testing and assessment (IATA), and quantitative AOP development for oxidative and mitochondrial toxicity.

Acknowledgement

This project was funded by the Research Council of Norway (RCN), grant no. RCN-315929 “EXPECT: In silico and experimental screening platform for characterizing environmental impact of industry development in the Arctic” (https://www.niva.no/en/projects/expect), the European Partnership for the Assessment of Risks from Chemicals (PARC) through European Union’s Horizon Europe research and innovation programme (Grant Agreement No 101057014, and supported by the NIVA Computational Toxicology Program, NCTP (https://www.niva.no/en/featured-pages/nctp, grant. No. RCN-342628).

AI disclosure

Artificial intelligence (AI) tools were used to support literature prioritization, review and AOP-Wiki page preparation in this work. AOP-helpFinder was used for automated literature mining, and ChatGPT (OpenAI) was used as an auxiliary tool for title and abstract screening, extraction of study metadata, and identification of potential weight-of-evidence indicators. AI-assisted outputs were used only to organize and prioritize information and were verified against the original sources by the authors before inclusion. Additional AI assistance was used for formatting, copy-editing, citation cross-checking, and harmonization of the AOP-Wiki pages. All scientific interpretations, weight-of-evidence judgments, final wording, and conclusions were determined and approved by the authors, who take full responsibility for the content and integrity of the work.

ROS are continuously formed during aerobic metabolism and can also be generated in response to environmental stressors. At controlled levels, ROS participate in redox signaling, whereas excessive ROS can disturb redox homeostasis and initiate oxidative stress (Schieber and Chandel, 2014; Sies et al., 2017). Lipid membranes are important targets of oxidative attack because phospholipids containing polyunsaturated fatty acids can undergo radical-driven peroxidation. Lipid peroxidation generates lipid hydroperoxides and secondary reactive aldehydes, including malondialdehyde and 4-hydroxy-2-nonenal, which can propagate oxidative injury and alter membrane-associated protein and organelle function (Ayala et al., 2014).

    AOP 331 was developed to represent the lipid peroxidation and cell injury/death-driven linear route within the broader ROS-growth AOP network. This route was selected because lipid peroxidation is a well-established consequence of oxidative stress and because mitochondrial membranes are central determinants of OXPHOS coupling. Peroxidative modification of mitochondrial membrane lipids can alter membrane fluidity, proton leak, respiratory control, and mitochondrial membrane potential, providing a mechanistically coherent bridge from oxidative stress to impaired ATP production (Murphy, 2009; Nicholls and Ferguson, 2013; Ouillon et al., 2021). ATP depletion is a well-established contributor to loss of cell viability because cellular survival depends on ATP-dependent ion gradients, membrane repair, protein turnover, stress-response pathways, and the execution of regulated death processes. Depletion of ATP can shift cells from adaptive responses to injury and death, and severe ATP loss can affect the mode of cell death (Leist et al., 1997; Bonora et al., 2012).

The AOP was also developed to take advantage of existing AOP-Wiki modularity. The upstream oxidative stress context is associated with AOP 478, while the OXPHOS-to-ATP KER is associated with AOP 263 (AOP-Wiki, 2026a,b; OECD, 2022). The cell injury/death KE is a highly reusable AOP-Wiki KE and appears across several established AOPs. AOP 17 explicitly includes oxidative stress leading to cell injury/death and also includes several KERs involving cell injury/death and neuroinflammation (AOP-Wiki, 2026e). AOP 48 includes mitochondrial dysfunction leading to cell injury/death in an excitotoxicity context (AOP-Wiki, 2026g). AOP 38 uses cell injury and cell death as key early tissue-level consequences of protein alkylation leading to fibrosis (AOP-Wiki, 2026f). AOPs 12 and 13 also use cell injury/death in neurodegeneration and synaptogenesis-related contexts (AOP-Wiki, 2026c,d).  These associations support the reuse of Event 55 as a generic, modular cellular KE downstream of multiple upstream stressors and upstream of multiple adverse outcomes.

AOP 331 was developed using the principles described in OECD AOP guidance, including modular description of KEs and KERs, reuse of existing AOP-Wiki content where appropriate, evidence evaluation using biological plausibility, empirical support, essentiality, and quantitative understanding, and clear description of the biological domain of applicability (OECD, 2018, 2021). The aim was to assemble a focused linear pathway from reusable AOP-Wiki elements rather than to create an isolated de novo pathway. This is important because AOP 331 is one branch of the broader ROS-growth AOP network and because its KEs overlap with oxidative stress, mitochondrial dysfunction, cellular energy metabolism, cell injury/death, and growth-related AOPs.

    Reuse of existing AOP-Wiki content was considered at the outset. AOP 478 was reviewed because it provides an AOP-Wiki precedent for oxidative stress as a central KE downstream of free radical generation and energy deposition. AOP 263 was reviewed because it provides an OECD-published downstream bioenergetics module in which decreased coupling of OXPHOS leads to decreased ATP pool and subsequently growth inhibition, although in AOP 263 the terminal cellular route proceeds through decreased cell proliferation rather than cell injury/death. AOPs 12, 13, 17, 38, and 48 were reviewed because they demonstrate repeated reuse of Event 55, 'Increase, Cell injury/death', across different biological contexts and provide support for treating cell injury/death as a modular KE that can connect distinct upstream mechanisms to downstream tissue or organism-level outcomes. AOP 296 was reviewed during development of the broader ROS-growth network to ensure that oxidative stress and macromolecular damage modules were harmonized with existing oxidative damage content, although AOP 331 specifically follows the lipid peroxidation and bioenergetic injury branch rather than the oxidative DNA damage branch.

    The evidence base was assembled through an AI-human hybrid workflow. First, search terms were developed for each KE, including KE names, synonyms, endpoint names, assay terms, taxa, and representative stressors. AOP-helpFinder was used to search PubMed for co-occurrence between key events and related biological concepts, and the exported outputs included PMIDs, titles, abstracts, and matched KE terms (Carvaillo et al., 2019; Jornod et al., 2022). The exported records were subjected to overlap analysis to remove redundant hits and to filter taxa-related or clearly irrelevant literature.

    Second, ChatGPT (OpenAI, San Francisco, CA, USA)-assisted screening was used as an auxiliary prioritization step. The LLM was used to pre-screen titles and abstracts, extract study metadata including stressor, species, biological system, dose or concentration, and exposure time, identify evidence types such as biological plausibility, empirical support, and essentiality, and flag weight-of-evidence indicators such as dose-response concordance, temporal concordance, incidence concordance, and intervention evidence. The LLM output was used to classify studies as high relevance, medium relevance, or low/not relevant. High-relevance studies were retrieved for full-text review, medium-relevance studies were reserved as supporting evidence, and low-relevance studies were documented as low priority or excluded.

    Third, full-text review and expert curation were used to verify all evidence before inclusion in the AOP. LLM-assisted full-text review was used only to organize candidate evidence; all extracted information was checked manually against the original text. Expert review was then used to populate KER evidence tables with methods, endpoints, results, weight-of-evidence category, and references. Final weight-of-evidence evaluation was performed by expert judgment using biological plausibility, empirical support, essentiality, quantitative understanding, and identification of evidence gaps. Thus, the development process combined text-mining and AI-assisted evidence handling with human expert verification and final decision-making.

    In parallel with this workflow, targeted searches were conducted to fill specific evidence gaps for ROS, oxidative stress, lipid peroxidation, mitochondrial membrane potential, OXPHOS coupling, ATP depletion, cytotoxicity, cell death, and growth inhibition. Studies were prioritized when they measured two or more KEs in the same biological system, reported dose or concentration and exposure time, or provided evidence relevant to dose-response, temporal, or incidence concordance. Mechanistic reviews and OECD reports were used primarily to support biological plausibility, while primary experimental studies were used to support empirical concordance wherever possible.

The overall weight of evidence supporting AOP 331 is considered moderate. Biological plausibility is high for all six KERs in the pathway. The upstream oxidative stress, lipid peroxidation, and OXPHOS uncoupling sequence follows a well-established mechanistic logic, and the connection from ATP depletion to cell injury/death is supported by the fundamental dependence of cellular survival on adequate energy supply. The cell injury/death-to-growth relationship is reinforced by the broad reuse of Event 55 (Increase, Cell injury/death) as a modular KE across endorsed AOPs 12, 13, 17, 38, and 48 (AOP-Wiki, 2026a-e). The OXPHOS-to-ATP module is directly associated with OECD-endorsed AOP 263 and contributes high biological plausibility and strong quantitative understanding for this segment (OECD, 2022; Song and Villeneuve, 2021). Empirical support is high for the ROS-to-oxidative-stress and oxidative-stress-to-lipid-peroxidation relationships, moderate for the lipid-peroxidation-to-OXPHOS link, and moderate to high for the ATP-depletion-to-cell-death and OXPHOS-to-ATP relationships. The cell death-to-growth relationship has moderate empirical support, as direct concurrent measurement of cell injury/death and organismal growth is less common across the available literature. Essentiality is rated moderate to high overall, with the strongest direct evidence for the AOP 263 bioenergetics segment. Quantitative understanding is highest for the OXPHOS-to-ATP KER and low to moderate elsewhere. The main uncertainties are the quantitative thresholds governing the lipid-peroxidation-to-OXPHOS transition, the severity-dependent mode of cell death triggered by ATP depletion, and the extent to which cell injury/death versus reduced proliferation drives growth impairment in specific biological contexts. AOP 331 is most appropriate for mechanistic interpretation of cytotoxic growth impairment caused by oxidative lipid damage, IATA development, and chemical prioritisation (OECD, 2018; Becker et al., 2015).

AOP 331 can support mechanistic interpretation of growth impairment caused by oxidative stressors that induce lipid peroxidation, mitochondrial bioenergetic dysfunction, ATP depletion, and cell injury/death. The AOP is particularly relevant for hazard identification and chemical prioritization when evidence indicates increased ROS or oxidative stress together with lipid peroxidation, mitochondrial membrane potential changes, reduced respiratory control, ATP depletion, cytotoxicity, or growth inhibition. The AOP may also support IATA development by linking upstream NAM endpoints, such as ROS assays, lipid peroxidation markers, mitochondrial membrane potential, oxygen consumption rate, ATP content, cytotoxicity assays, and organismal growth measurements.

AOP 331 can support chemical grouping and read-across for stressors that share evidence of oxidative lipid damage, mitochondrial bioenergetic impairment, and ATP-associated cell injury. Because oxidative stress and lipid peroxidation are not chemical-specific, this AOP should not be used as a stand-alone basis for regulatory decisions. Instead, it should be applied as part of a weight-of-evidence framework that considers stressor mode of action, exposure context, assay specificity, taxonomic relevance, and concordance across multiple KEs. The AOP also highlights method-development needs, particularly standardized assays for lipid peroxidation, OXPHOS coupling, ATP depletion, and cell injury/death endpoints that can be connected quantitatively to apical growth outcomes.

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