Reactive oxygen species leading to growth inhibition via oxidative DNA damage 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 325) describes a linear pathway by which excessive reactive oxygen species (ROS) can lead to decreased organismal growth through oxidative DNA damage and cell injury/death. The pathway is one branch of the broader ROS-growth AOP network and represents a DNA damage- and cytotoxicity-driven route from a conserved early oxidative perturbation to an adverse outcome of regulatory relevance. In this AOP, increased ROS (Event 1115) is treated operationally as the earliest common measurable initiating perturbation shared by diverse stressors that increase intracellular oxidant burden. Increased ROS leads to oxidative stress (Event 1392), which promotes oxidative DNA damage (Event 1634). When oxidative DNA lesions are not repaired correctly or efficiently, inadequate DNA repair (Event 155) and accumulation of DNA strand breaks (Event 1635) can occur. Severe or persistent DNA strand breaks activate DNA damage response pathways and can lead to increased cell injury/death (Event 55). At higher levels of biological organization, excessive cell loss reduces tissue growth capacity and contributes to decreased growth (Event 1521).

    The AOP reuses established AOP-Wiki content and is associated with several existing AOPs. The oxidative DNA damage module is closely aligned with AOP 296, which describes oxidative DNA damage leading to mutations and chromosomal aberrations (OECD, 2023). The upstream radiation/ROS context is informed by AOP 478, which includes ROS-mediated DNA damage following energy deposition. The cell injury/death event is a broadly reused KE in AOPs 12, 13, 17, 38, and 48, which collectively demonstrate that cell injury/death is a modular downstream consequence of diverse molecular insults. The adverse outcome of decreased growth is shared with AOP 263, an OECD-endorsed AOP describing growth inhibition via reduced cell proliferation following energetic impairment (OECD, 2022).

Biological plausibility is high for most KERs in the pathway because ROS-mediated oxidative stress, oxidative DNA damage, DNA repair failure, DNA strand break formation, DNA damage response activation, and cell death are well-established and highly conserved biological processes (Cooke et al., 2003; Cuddihy and O'Connell, 2003; Sies et al., 2017). Empirical support is strongest for the early oxidative stress and DNA damage KERs and moderate to high for the downstream progression from DNA strand breaks to cell injury/death and growth impairment. This AOP is relevant to environmental and human health risk assessment, particularly for stressors such as hydrogen peroxide, paraquat, metals, ionizing radiation, ultraviolet radiation, and silver-based materials that can increase ROS and DNA damage.

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 are also generated in response to environmental stressors. At controlled levels, ROS participate in redox signaling, whereas excessive ROS can disturb redox homeostasis and initiate oxidative damage to cellular macromolecules (Schieber and Chandel, 2014; Sies et al., 2017). DNA is a major target of oxidative attack. Oxidative DNA lesions such as 8-oxo-2'-deoxyguanosine and other oxidized bases can arise endogenously or following toxic insult, and these lesions may contribute to mutation, strand break formation, activation of DNA damage responses, and cell death if they are not repaired correctly or efficiently (Cooke et al., 2003; OECD, 2023).

    AOP 325 was developed to represent the cell injury/death-driven linear route within the broader ROS-growth AOP network. The route was selected because oxidative DNA damage is a well-established consequence of oxidative stress and because downstream events such as inadequate DNA repair, DNA strand breaks, and cell injury/death provide a mechanistically coherent bridge between molecular damage and decreased organismal growth. This pathway complements AOP 324, which emphasizes cell cycle disruption and reduced cell proliferation as the downstream route from DNA damage to growth impairment. AOP 325 instead captures the alternative but biologically connected route in which unrepaired or severe DNA damage contributes to cell loss, thereby reducing growth capacity at the organ or organism level.

    A key design principle was reuse of existing AOP-Wiki content. AOP 296 provides the most direct precedent for the oxidative DNA damage portion of the pathway because it treats oxidative DNA damage as an initiating event that can overwhelm repair processes and lead to strand breaks, mutation, and chromosomal aberrations (OECD, 2023). AOP 478 provides an associated radiation-relevant context in which energy deposition can generate ROS and induce DNA damage. AOP 17 includes oxidative stress and cell injury/death in a neurotoxicity context, and AOPs 12, 13, and 48 use cell injury/death as a key event in neurodevelopmental or neurodegenerative pathways. AOP 38 includes cell injury/death as an early key event following protein alkylation in liver fibrosis, demonstrating the modularity of this event beyond nervous system contexts. AOP 263 provides the shared adverse outcome of decreased growth and supports the regulatory relevance and broad taxonomic applicability of growth impairment as an apical endpoint (OECD, 2022).

Development of AOP 325 followed 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; OECD, 2021). Existing AOP-Wiki entries and OECD-endorsed AOPs were reviewed to identify reusable KEs, KERs, and evidence summaries. This reuse strategy was important because the pathway is not intended to redefine established biology; rather, it links reusable oxidative stress, DNA damage, cell injury/death, and growth endpoints into a focused linear route suitable for incorporation into the ROS-growth AOP network.

    The evidence base was assembled through a structured AI-human hybrid workflow. First, event-specific search terms were developed for each KE, including KE names, synonyms, endpoint terms, assay terms, taxa, and species. These terms were used in AOP-helpFinder to search PubMed for co-occurrence of KE-related concepts and to generate an initial evidence pool containing PMIDs, titles, abstracts, and matched KE terms (Carvaillo et al., 2019; Jornod et al., 2022). The AOP-helpFinder output was exported and subjected to overlap analysis to remove redundant hits and to filter literature that was clearly unrelated to the biological scope of the AOP.

In the second phase, a ChatGPT (GPT-4, OpenAI, San Francisco, CA, USA) was used as an auxiliary screening tool for title and abstract pre-screening. The large language model (LLM)-assisted step extracted study metadata such as stressor, species, biological system, dose or concentration, and exposure duration; identified the type of evidence represented by each study, including biological plausibility, empirical support, and essentiality; and flagged potential weight-of-evidence indicators such as dose-response concordance, temporal concordance, incidence concordance, and intervention or rescue evidence. The LLM output was used only to prioritize and organize the literature. It did not replace expert judgment.

    High-relevance records were retrieved for full-text review, whereas medium-relevance records were reserved as supporting evidence and low-relevance records were documented as excluded or low-priority. A second LLM-assisted full-text step was used to organize information from retrieved papers, but all LLM outputs were checked manually against the original article text. In the final phase, domain experts curated the evidence, populated KER evidence tables, assigned weight-of-evidence confidence levels, and identified uncertainties, inconsistencies, and evidence gaps. This workflow combined the efficiency of text-mining and AI-assisted screening with manual expert review, thereby improving transparency while preserving expert control over interpretation and final evidence evaluation.

Targeted literature searches were also performed to fill specific gaps. Searches focused on combinations of terms for reactive oxygen species, oxidative stress, oxidative DNA damage, DNA repair, DNA strand breaks, DNA damage response, cell injury, cell death, apoptosis, cytotoxicity, growth inhibition, paraquat, hydrogen peroxide, silver, ionizing radiation, ultraviolet radiation, fish embryos, algae, copepods, mollusks, and AOP. Studies were prioritized when they measured two or more KEs in the same biological system, reported exposure time and dose or concentration, or provided information relevant to dose-response, temporal, or incidence concordance. Mechanistic reviews and OECD reports were used to support biological plausibility, whereas primary experimental studies were used to support empirical concordance wherever possible (Cooke et al., 2003; Cuddihy and O'Connell, 2003; Qian et al., 2009; Hlavová et al., 2011; Han et al., 2014; Won and Lee, 2014; Quevedo et al., 2021; OECD, 2023).

The overall weight of evidence supporting AOP 325 is considered moderate. Biological plausibility is high for all six KERs in the pathway. The mechanistic connections between ROS accumulation, oxidative stress, oxidative DNA damage, inadequate DNA repair, DNA strand break formation, activation of DNA damage response pathways, and increased cell injury/death are well established, and the final link from cell injury/death to decreased growth is biologically coherent and supported by the wide reuse of Event 55 (Increase, Cell injury/death) as a modular KE in AOPs 12, 13, 17, 38, and 48 (AOP-Wiki, 2026a-e). Empirical support is high for the upstream ROS-to-oxidative-stress and oxidative-stressàDNA-damage relationships, moderate to high for the DNA strand breakàcell death transition, and moderate for the cell deathàgrowth relationship, where direct co-measurement of cell injury/death and organismal growth in the same study is less common. Essentiality of the KEs is rated moderate for most events, reflecting that upstream perturbation consistently reduces downstream damage outcomes but that alternative pathways can contribute independently to both cell death and growth inhibition. Quantitative understanding is low to moderate across most KERs, particularly for the threshold-dependent relationship between DNA strand breaks and cell death and for the translation of cell death to organismal growth impairment. The main uncertainties are the conditional nature of the DNA strand break-to-cell death relationship (which depends strongly on repair capacity, cell type, p53 status, and exposure severity), the possibility that growth impairment arises from reduced proliferation as much as from overt cell loss, and the multifactorial character of growth as an apical endpoint. AOP 325 is currently most suitable for qualitative use in mechanistic interpretation of cytotoxic growth impairment, hazard identification, and support for integrated testing and assessment strategies (OECD, 2018; Becker et al., 2015).

AOP 325 can support mechanistic interpretation of toxicity data when evidence indicates that a stressor increases ROS or oxidative stress and produces DNA damage, cytotoxicity, and growth impairment. The AOP is particularly useful for evaluating stressors such as radiation, redox-active chemicals, metals, and nanoparticles that may act through oxidative DNA damage. It can also support chemical prioritization and screening by linking early oxidative stress and DNA damage assays to potential effects on growth.

    The AOP is relevant to integrated approaches to testing and assessment because many of its KEs can be measured using established assays. ROS can be measured using DCFH-DA, DHE, MitoSOX, or electron spin resonance; oxidative stress can be assessed through antioxidant enzyme activity, GSH/GSSG ratio, or Nrf2/ARE reporter assays; oxidative DNA damage can be assessed using 8-oxo-dG measurements or enzyme-modified comet assays; DNA strand breaks can be assessed using alkaline comet assays or gamma-H2AX staining; and cell injury/death can be measured using viability, LDH release, Annexin V/PI staining, caspase activity, or TUNEL assays. The final AO, decreased growth, is directly relevant to standardized ecotoxicological endpoints measured in algae, aquatic invertebrates, fish, amphibians, and plants.

    The AOP also identifies important limitations. Quantitative prediction of growth from upstream DNA damage or cell death remains underdeveloped, and cell injury/death is not the only route by which ROS can impair growth. The AOP should therefore be applied as part of a broader weight-of-evidence evaluation and, where relevant, considered together with other ROS-growth AOPs that describe cell cycle disruption, reduced proliferation, mitochondrial dysfunction, lipid peroxidation, and protein oxidation pathways.

Abbott, B. D., Harris, M. W., & Birnbaum, L. S. (1995). Cell death in rat and mouse embryos exposed to methanol in whole embryo culture: Evaluation of the role of the p53 tumor suppressor gene. Teratogenesis, Carcinogenesis, and Mutagenesis, 15, 147-169.

AOP-Wiki. (2026a). AOP 12: Chronic binding of antagonist to N-methyl-D-aspartate receptors during brain development induces impairment of learning and memory abilities. AOP-Wiki. Accessed 14 May 2026.

AOP-Wiki. (2026b). AOP 13: Chronic binding of antagonist to N-methyl-D-aspartate receptors during brain development leading to impairment of learning and memory. AOP-Wiki. Accessed 14 May 2026.

AOP-Wiki. (2026c). AOP 17: Binding of electrophilic chemicals to SH-/selenoproteins involved in protection against oxidative stress leading to impairment of learning and memory. AOP-Wiki. Accessed 14 May 2026.

AOP-Wiki. (2026d). AOP 38: Protein alkylation leading to liver fibrosis. AOP-Wiki. Accessed 14 May 2026.

AOP-Wiki. (2026e). AOP 48: Binding of agonists to ionotropic glutamate receptors in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment. AOP-Wiki. Accessed 14 May 2026.

AOP-Wiki. (2026f). AOP 478: Deposition of energy leading to occurrence of cataracts. AOP-Wiki. Accessed 14 May 2026.

Carvaillo, J.-C., Barouki, R., Coumoul, X., & Audouze, K. (2019). Linking bisphenol S to adverse outcome pathways using a combined text mining and systems biology approach. Environmental Health Perspectives, 127(4), 047005. https://doi.org/10.1289/EHP4200

Conlon, I., & Raff, M. (1999). Size control in animal development. Cell, 96(2), 235-244. https://doi.org/10.1016/S0092-8674(00)80563-2

Cooke, M. S., Evans, M. D., Dizdaroglu, M., & Lunec, J. (2003). Oxidative DNA damage: Mechanisms, mutation, and disease. FASEB Journal, 17(10), 1195-1214. https://doi.org/10.1096/fj.02-0752rev

Cuddihy, A. R., & O'Connell, M. J. (2003). Cell-cycle responses to DNA damage in G2. International Review of Cytology, 222, 99-140. https://doi.org/10.1016/S0074-7696(02)22013-6

Han, J., Won, E.-J., Lee, B.-Y., Hwang, U.-K., Kim, I.-C., Yim, J. H., Leung, K. M. Y., Lee, Y. S., & Lee, J.-S. (2014). Gamma rays induce DNA damage and oxidative stress associated with impaired growth and reproduction in the copepod Tigriopus japonicus. Aquatic Toxicology, 152, 264-272. https://doi.org/10.1016/j.aquatox.2014.04.005

Hlavová, M., Čížková, M., Vítová, M., Bišová, K., & Zachleder, V. (2011). DNA damage during G2 phase does not affect cell cycle progression of the green alga Scenedesmus quadricauda. PLoS ONE, 6(5), e19626. https://doi.org/10.1371/journal.pone.0019626

Jornod, F., Jaylet, T., Blaha, L., Sarigiannis, D., Tamisier, L., & Audouze, K. (2022). AOP-helpFinder webserver: A tool for comprehensive analysis of the literature to support adverse outcome pathways development. Bioinformatics, 38(4), 1173-1175. https://doi.org/10.1093/bioinformatics/btab750

Leist, M., Single, B., Castoldi, A. F., Kuhnle, S., & Nicotera, P. (1997). Intracellular adenosine triphosphate concentration: A switch in the decision between apoptosis and necrosis. Journal of Experimental Medicine, 185(8), 1481-1486. https://doi.org/10.1084/jem.185.8.1481

Melo, K. M., Oliveira, R., Grisolia, C. K., Domingues, I., Pieczarka, J. C., de Souza Filho, J., & Nagamachi, C. Y. (2015). Short-term exposure to low doses of rotenone induces developmental, biochemical, behavioral, and histological changes in fish. Environmental Science and Pollution Research, 22(18), 13926-13938. https://doi.org/10.1007/s11356-015-4626-0

Mitchelmore, C. L., & Chipman, J. K. (1998). DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 399(2), 135-147. https://doi.org/10.1016/S0027-5107(97)00252-2

Nicotera, P., Leist, M., & Ferrando-May, E. (1998). Intracellular ATP, a switch in the decision between apoptosis and necrosis. Toxicology Letters, 102-103, 139-142. https://doi.org/10.1016/S0378-4274(98)00298-7

O'Connell, M. J., Walworth, N. C., & Carr, A. M. (2000). The G2-phase DNA-damage checkpoint. Trends in Cell Biology, 10(7), 296-303. https://doi.org/10.1016/S0962-8924(00)01773-6

OECD. (2018). Users' Handbook Supplement to the Guidance Document for Developing and Assessing Adverse Outcome Pathways. OECD Series on Adverse Outcome Pathways No. 1. OECD Publishing, Paris.

OECD. (2021). Guidance Document for the Scientific Review of Adverse Outcome Pathways. OECD Series on Testing and Assessment No. 344. OECD Publishing, Paris.

OECD. (2022). Uncoupling of Oxidative Phosphorylation Leading to Growth Inhibition via Decreased Cell Proliferation. OECD Series on Adverse Outcome Pathways No. 28. OECD Publishing, Paris.

OECD. (2023). Oxidative DNA damage leading to chromosomal aberrations and mutations. OECD Series on Adverse Outcome Pathways, No. 29. OECD Publishing, Paris. https://doi.org/10.1787/399d2c34-en

Qian, H., Chen, W., Sun, L., Jin, Y., Liu, W., & Fu, Z. (2009). Inhibitory effects of paraquat on photosynthesis and the response to oxidative stress in Chlorella vulgaris. Ecotoxicology, 18(5), 537-543. https://doi.org/10.1007/s10646-009-0311-8

Quevedo, A. C., Lynch, I., & Valsami-Jones, E. (2021). Cellular repair mechanisms triggered by exposure to silver nanoparticles and ionic silver in embryonic zebrafish cells. Environmental Science: Nano, 8(9), 2507-2522. https://doi.org/10.1039/D1EN00422K

Roos, W. P., & Kaina, B. (2006). DNA damage-induced cell death by apoptosis. Trends in Molecular Medicine, 12(9), 440-450. https://doi.org/10.1016/j.molmed.2006.07.007

Schieber, M., & Chandel, N. S. (2014). ROS function in redox signaling and oxidative stress. Current Biology, 24(10), R453-R462. https://doi.org/10.1016/j.cub.2014.03.034

Sies, H., Berndt, C., & Jones, D. P. (2017). Oxidative stress. Annual Review of Biochemistry, 86, 715-748. https://doi.org/10.1146/annurev-biochem-061516-045037

Won, E.-J., & Lee, J.-S. (2014). Gamma radiation induces growth retardation, impaired egg production, and oxidative stress in the marine copepod Paracyclopina nana. Aquatic Toxicology, 150, 17-26. https://doi.org/10.1016/j.aquatox.2014.02.010