Reactive oxygen species leading to growth inhibition via protein oxidation 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 333) describes a linear route by which increased reactive oxygen species (ROS) can lead to decreased organismal growth through oxidative stress-mediated protein oxidation, mitochondrial bioenergetic impairment, ATP depletion, and increased cell injury/death. 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. When oxidant production exceeds antioxidant and repair capacity, proteins become oxidatively modified through processes such as carbonylation, thiol oxidation, glutathionylation, nitration, fragmentation, aggregation, or altered degradation. Oxidative modification of proteins involved in mitochondrial electron transport, ATP synthase activity, substrate transport, or maintenance of mitochondrial membrane potential can impair coupling of oxidative phosphorylation (OXPHOS). Decreased OXPHOS coupling reduces the cellular ATP pool. ATP depletion compromises ion homeostasis, membrane integrity, stress-response capacity, biosynthesis, and the execution of regulated cell death pathways, thereby increasing cell injury/death. Increased loss of viable cells can reduce tissue or organismal growth, particularly in developing, rapidly growing, or metabolically active systems.

    AOP 333 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 energy deposition generates free radicals and oxidative stress and includes oxidative damage to proteins as a downstream consequence (AOP-Wiki, 2026a). The relationship from decreased coupling of OXPHOS to decreased ATP pool is associated with AOP 263, an OECD-published AOP that links OXPHOS uncoupling to growth inhibition through ATP depletion and reduced cell proliferation (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021). AOP 333 differs from AOP 332 by routing ATP depletion through increased cell injury/death rather than through decreased cell proliferation. The cellular injury/death module reuses the broadly shared AOP-Wiki KE 'Increase, Cell injury/death', which occurs in AOPs 12, 13, 17, 38, and 48 in neurotoxicity, oxidative stress, fibrosis, and excitotoxicity contexts (AOP-Wiki, 2026c-g). The AOP is relevant to environmental and human health contexts because ROS generation, oxidative stress responses, protein oxidation, mitochondrial ATP production, cell viability, and growth are broadly conserved among aerobic eukaryotes. The AOP can support mechanistic interpretation of oxidative stress-mediated growth impairment, assay selection, chemical prioritization, integrated approaches to testing and assessment (IATA), and future 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). Proteins are major targets of oxidative attack because amino acid side chains, thiol groups, metal centers, and prosthetic groups can undergo oxidative modification. Protein oxidation can reduce enzyme activity, alter protein-protein interactions, impair folding, increase aggregation, disrupt degradation by proteasomal and lysosomal systems, and contribute to cellular dysfunction (Dalle-Donne et al., 2006).

    AOP 333 was developed to represent the protein oxidation and cell injury/death-driven linear route within the broader ROS-growth AOP network. This route was selected because protein oxidation is a well-established consequence of oxidative stress and because mitochondrial proteins are central determinants of OXPHOS coupling and ATP production. Oxidative modification of respiratory-chain proteins, ATP synthase subunits, mitochondrial carriers, or proteins involved in maintaining mitochondrial membrane potential can reduce respiratory efficiency and ATP synthesis (Murphy, 2009; Nicholls and Ferguson, 2013; Sokolov et al., 2019). ATP depletion is an established contributor to loss of cell viability because cell survival depends on ATP-dependent ion gradients, membrane repair, stress responses, proteostasis, and execution of regulated cell death pathways. Severe ATP depletion can shift cellular outcomes toward irreversible injury or necrosis, whereas less severe depletion may permit apoptosis or adaptive responses (Leist et al., 1997; Bonora et al., 2012).

The AOP was designed to maximize reuse of existing AOP-Wiki content. AOP 478 was reviewed because it provides a curated AOP-Wiki context for oxidative stress downstream of free radical generation and includes oxidative molecular damage, including modified proteins, as a relevant consequence of oxidative stress (AOP-Wiki, 2026a). AOP 263 was used to anchor the downstream mitochondrial bioenergetic segment because it provides an OECD-published, well-supported module connecting decreased coupling of OXPHOS with decreased ATP pool and decreased growth-related outcomes (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021). AOPs 12, 13, 17, 38, and 48 were reviewed because they reuse the KE 'Increase, Cell injury/death' and provide evidence that cell injury/death is a generic, reusable cellular response to diverse upstream perturbations. In particular, AOP 17 describes oxidative stress-related developmental neurotoxicity that includes cell injury/death, AOP 38 uses cell injury/death as an early response to protein alkylation in liver fibrosis, and AOP 48 includes mitochondrial dysfunction leading to cell injury/death in an excitotoxicity context (AOP-Wiki, 2026e-g).

AOP 333 was developed using the principles described in OECD AOP guidance, including modular description of KEs and KERs, evidence evaluation using biological plausibility, empirical support, essentiality, and quantitative understanding, and clear description of the biological domain of applicability (OECD, 2018, 2021). The development approach combined reuse of existing AOP-Wiki content, targeted literature review, and an AI-human hybrid evidence workflow. The objective was to define a focused linear AOP within the broader ROS-growth AOP network rather than to create an isolated de novo pathway.

    The evidence search began with development of event-specific search terms for each KE, including KE names, synonyms, endpoint terms, assay names, stressor terms, taxa, and species names. These terms were used in AOP-helpFinder to search PubMed for co-occurrence of KEs and related mechanistic concepts (Carvaillo et al., 2019; Jornod et al., 2022). AOP-helpFinder outputs, including PMIDs, titles, abstracts, and matched KE terms, were exported and subjected to overlap analysis to remove redundant hits and filter taxa- or endpoint-irrelevant literature.

    The second phase used ChatGPT (OpenAI, San Francisco, CA, USA)-assisted screening to prioritize abstracts and full-text records. The LLM was used as an auxiliary tool to extract study metadata, including stressor, species, biological system, dose or concentration, and exposure time; to classify evidence type, including biological plausibility, empirical support, and essentiality; and to identify weight-of-evidence indicators, including dose-response concordance, temporal concordance, incidence concordance, and intervention or rescue evidence. Studies were categorized as high, medium, or low priority. High-priority studies were retrieved for full-text review, medium-priority studies were reserved as supporting evidence, and low-priority studies were documented but not carried forward for detailed curation.

The final phase consisted of manual expert review and curation. Expert review verified LLM outputs against the full text, extracted evidence into KER evidence tables, and assigned weight-of-evidence calls for biological plausibility, empirical support, essentiality, and quantitative understanding. Targeted manual searches were performed to fill gaps for protein oxidation, mitochondrial bioenergetics, ATP depletion, cell injury/death, and growth outcomes. Studies were prioritized when they measured two or more KEs in the same biological system, reported dose and time information, or supported temporal, dose-response, incidence, or intervention concordance. Mechanistic reviews and OECD reports were used to support biological plausibility where relationships are widely established, whereas primary experimental studies were prioritized for empirical support where available.

The overall weight of evidence supporting AOP 333 is considered moderate. Biological plausibility is high for all six KERs in the pathway. The mechanistic connections between oxidative stress, protein oxidation, impaired mitochondrial OXPHOS coupling, ATP depletion, cell injury/death, and decreased growth are individually well supported, and the AOP draws on conserved biological processes broadly applicable across aerobic eukaryotes. The central OXPHOS-to-ATP segment is directly associated with OECD-endorsed AOP 263, providing strong mechanistic and quantitative support for this portion of the pathway (OECD, 2022; Song and Villeneuve, 2021). The cell injury/death KE (Event 55) is a widely reused and modular AOP-Wiki element present in endorsed AOPs 12, 13, 17, 38, and 48, reinforcing the credibility of its use as a downstream consequence of severe energetic failure (AOP-Wiki, 2026a-e). Empirical support is high for the ROS-to-oxidative-stress and oxidative-stress-to-protein-oxidation relationships and moderate for the protein-oxidation-to-OXPHOS transition, where supporting evidence is observational and cross-stressor rather than from controlled selective-inhibition studies. The ATP-depletion-to-cell-death and cell-death-to-growth KERs have moderate empirical support. Essentiality is high for the OXPHOS-to-ATP relationship and moderate for the remaining KEs. Quantitative understanding is strongest for the OXPHOS-to-ATP KER and low to moderate elsewhere, reflecting the difficulty of predicting organism-level growth outcomes from upstream molecular damage endpoints. The main uncertainties are the causal versus correlational character of the protein oxidation-OXPHOS association, the ATP threshold dependence of cell death mode and severity, and the multifactorial nature of organismal growth as an apical endpoint. AOP 333 is currently most suitable for qualitative and semi-quantitative use in mechanistic interpretation, hazard identification, and support for integrated testing and assessment strategies (OECD, 2018; Becker et al., 2015).

AOP 333 can support mechanistic interpretation of growth impairment caused by oxidative stressors that induce protein oxidation, 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 protein carbonylation, redox proteomic signatures, 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, oxidative stress biomarkers, protein carbonyl assays, redox proteomics, mitochondrial membrane potential, oxygen consumption rate, ATP content, cytotoxicity assays, and organismal growth measurements.

AOP 333 can support chemical grouping and read-across for stressors that share evidence of oxidative protein damage, mitochondrial bioenergetic impairment, and ATP-associated cell injury. Because oxidative stress and protein oxidation 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 protein oxidation, OXPHOS coupling, ATP depletion, and cell injury/death endpoints that can be connected quantitatively to apical growth outcomes.

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