Increase, ROS leads to Increase, Oxidative Stress

This KER describes the causal and predictive relationship by which an increase in reactive oxygen species leads to oxidative stress. ROS include superoxide, hydrogen peroxide, hydroxyl radical and secondary oxygen-derived reactive products. At low or transient levels, ROS can participate in normal cell signaling. However, when ROS production, flux or local concentration exceeds the capacity of enzymatic and non-enzymatic antioxidant defenses, the redox balance of the biological system shifts toward an oxidizing state, producing oxidative stress (Schieber and Chandel, 2014; Sies et al., 2017).

    The downstream KE, oxidative stress, is not identical to increased ROS. Rather, it represents a systems-level imbalance between pro-oxidant pressure and antioxidant or repair capacity. The KER therefore depends not only on the magnitude of ROS increase, but also on the duration, localization and chemical identity of the ROS, the capacity of scavenging systems such as glutathione, superoxide dismutase, catalase and glutathione peroxidases, and the ability of the cell or organism to activate adaptive redox responses such as NRF2 signaling (Halliwell and Gutteridge, 2015; Griendling et al., 2016; Sies et al., 2017).

Within the ROS-growth AOP network, Relationship 2009 functions as a shared upstream KER. It connects the early measurable perturbation of increased ROS to the central hub event of oxidative stress, from which downstream AOP branches proceed through oxidative DNA damage, lipid peroxidation, protein oxidation, mitochondrial dysfunction, ATP depletion, altered cell proliferation, cell injury/death and decreased growth. This KER should remain modular and stressor-agnostic; stressor-specific mechanisms of ROS generation should be described in MIE or stressor sections where appropriate.

Evidence for this KER was assembled from three complementary sources. First, existing AOP-Wiki content was reviewed, including the current Relationship 2009 page and the corresponding KE pages for Event 1115 and Event 1392. These pages define the upstream and downstream events and document existing applicability and evidence summaries (AOP-Wiki, 2026a, 2026b, 2026c). Second, the ROS-growth literature review and data-mining exercise was used to identify studies in which ROS and oxidative stress or antioxidant-response endpoints were measured in the same biological system following exposure to relevant stressors. Third, targeted searches of mechanistic reviews and primary studies were used to support biological plausibility, empirical concordance and domain-of-applicability statements.

    The evidence search strategy followed the AI-human hybrid workflow used for the ROS-growth AOPN. Search terms were developed for the upstream and downstream KEs, including “reactive oxygen species”, “ROS”, “oxidative stress”, “antioxidant enzyme”, “superoxide dismutase”, “catalase”, “glutathione peroxidase”, “glutathione”, “Nrf2”, “redox imbalance”, and representative stressor terms such as paraquat, hydrogen peroxide, copper, cadmium, chlorothalonil, radiation, hypoxia/reoxygenation and pathogen infection. AOP-helpFinder and targeted database searches were used to identify candidate studies, and ChatGPT (OpenAI, San Francisco, CA, USA)-assisted screening was used only for preliminary prioritization and metadata extraction. Final inclusion, interpretation and weight-of-evidence calls were made by expert review of the original sources.

Studies were prioritized when they measured both upstream and downstream events, reported dose or concentration and exposure duration, used biologically interpretable oxidative stress endpoints, and provided information relevant to dose-response, temporal or incidence concordance. Endpoints considered supportive of the downstream KE included antioxidant enzyme induction or depletion, GSH/GSSG changes, NRF2/ARE pathway activation, lipid peroxidation, and broader redox imbalance. Direct ROS measurements were given greater weight than studies in which ROS production was inferred solely from the stressor mode of action.

Quantitative understanding of this KER is low to moderate. The qualitative relationship is well established: oxidative stress occurs when ROS production or flux exceeds antioxidant and repair capacity. However, a universal quantitative threshold for ROS leading to oxidative stress cannot be defined because the relationship depends strongly on ROS species, subcellular localization, measurement method, antioxidant capacity, exposure duration, organism, cell type and co-stressors (Kalyanaraman et al., 2012; Griendling et al., 2016; Sies et al., 2017).

This KER is broadly applicable to aerobic eukaryotic systems in which ROS production and antioxidant buffering can be measured. The current AOP-Wiki relationship page identifies human, mouse and rat with high evidence, but the ROS-growth evidence base supports extension to algae, fish, crustaceans, mollusks and other organisms relevant to environmental toxicology (AOP-Wiki, 2026a). The relationship is expected to be conserved because it is based on redox chemistry and conserved antioxidant-defense systems rather than on a taxon-specific receptor or signaling pathway.

The applicability domain should nevertheless be bounded by biological context and measurement feasibility. This KER is most relevant when the upstream KE is a measurable increase in ROS and the downstream KE is a measurable redox imbalance or antioxidant-response state rather than a distal oxidative damage endpoint alone. In organisms or compartments where ROS cannot be measured directly, evidence may rely on antioxidant-response or oxidative damage biomarkers, but these should be interpreted as indirect support. Applicability is strongest when ROS and oxidative stress endpoints are measured in the same system under the same exposure conditions.

AOP-Wiki. 2026a. Relationship 2009: Increase, ROS leads to Increase, Oxidative stress. AOP-Wiki. Available at: https://aopwiki.org/relationships/2009. Accessed 14 May 2026.

AOP-Wiki. 2026b. Event 1115: Increase, Reactive oxygen species. AOP-Wiki. Available at: https://aopwiki.org/events/1115. Accessed 14 May 2026.

AOP-Wiki. 2026c. Event 1392: Increase, Oxidative stress. AOP-Wiki. Available at: https://aopwiki.org/events/1392. Accessed 14 May 2026.

Barata C, Varo I, Navarro JC, Arun S, Porte C. 2005. Antioxidant enzyme activities and lipid peroxidation in the freshwater cladoceran Daphnia magna exposed to redox cycling compounds. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 140(2):175-186. https://doi.org/10.1016/j.cca.2005.01.013.

Bedard K, Krause KH. 2007. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiological Reviews 87(1):245-313. https://doi.org/10.1152/physrev.00044.2005.

Dickinson BC, Chang CJ. 2011. Chemistry and biology of reactive oxygen species in signaling or stress responses. Nature Chemical Biology 7(8):504-511. https://doi.org/10.1038/nchembio.607.

Esperanza M, Cid A, Herrero C, Rioboo C. 2015. Acute effects of a prooxidant herbicide on the microalga Chlamydomonas reinhardtii: screening cytotoxicity and genotoxicity endpoints. Aquatic Toxicology 165:210-221. https://doi.org/10.1016/j.aquatox.2015.06.004.

Gao J, Liu M, Guo H, Zhu K, Liu B, Liu B, Zhang N, Sun X, Jiang S, Zhang D. 2022. ROS induced by Streptococcus agalactiae activate inflammatory responses via the TNF-alpha/NF-kappaB signaling pathway in golden pompano Trachinotus ovatus (Linnaeus, 1758). Antioxidants 11(9):1809. https://doi.org/10.3390/antiox11091809.

Griendling KK, Touyz RM, Zweier JL, Dikalov S, Chilian W, Chen YR, Harrison DG, Bhatnagar A. 2016. Measurement of reactive oxygen species, reactive nitrogen species, and redox-dependent signaling in the cardiovascular system: a scientific statement from the American Heart Association. Circulation Research 119(5):e39-e75. https://doi.org/10.1161/RES.0000000000000110.

Halliwell B, Gutteridge JMC. 2015. Free Radicals in Biology and Medicine. 5th ed. Oxford: Oxford University Press.

Haque MN, Eom HJ, Nam SE, Shin YK, Rhee JS. 2019. Chlorothalonil induces oxidative stress and reduces enzymatic activities of Na+/K+-ATPase and acetylcholinesterase in gill tissues of marine bivalves. PLoS ONE 14(4):e0214236. https://doi.org/10.1371/journal.pone.0214236.

Jian Z, Guo H, Liu H, Cui H, Fang J, Zuo Z, Deng J, Li Y, Wang X, Zhao L. 2020. Oxidative stress, apoptosis and inflammatory responses involved in copper-induced pulmonary toxicity in mice. Aging 12(17):16867-16886. https://doi.org/10.18632/aging.103585.

Kalyanaraman B, Darley-Usmar V, Davies KJA, Dennery PA, Forman HJ, Grisham MB, Mann GE, Moore K, Roberts LJ II, Ischiropoulos H. 2012. Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radical Biology and Medicine 52(1):1-6. https://doi.org/10.1016/j.freeradbiomed.2011.09.030.

Murphy MP. 2009. How mitochondria produce reactive oxygen species. Biochemical Journal 417(1):1-13. https://doi.org/10.1042/BJ20081386.

Ouillon N, Sokolov EP, Otto S, Rehder G, Sokolova IM. 2021. Effects of variable oxygen regimes on mitochondrial bioenergetics and reactive oxygen species production in a marine bivalve, Mya arenaria. Journal of Experimental Biology 224(4):jeb237156. https://doi.org/10.1242/jeb.237156.

Pan YX, Luo Z, Zhuo MQ, Wei CC, Chen GH, Song YF. 2018. Oxidative stress and mitochondrial dysfunction mediated Cd-induced hepatic lipid accumulation in zebrafish Danio rerio. Aquatic Toxicology 199:12-20. https://doi.org/10.1016/j.aquatox.2018.03.017.

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.

Schieber M, Chandel NS. 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 DP. 2017. Oxidative stress. Annual Review of Biochemistry 86:715-748. https://doi.org/10.1146/annurev-biochem-061516-045037.

Tseng YC, Chen RD, Lucassen M, Schmidt MM, Dringen R, Abele D, Hwang PP. 2011. Exploring uncoupling proteins and antioxidant mechanisms under acute cold exposure in brains of fish. PLoS ONE 6(3):e18180. https://doi.org/10.1371/journal.pone.0018180.

Wardman P. 2007. Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects. Free Radical Biology and Medicine 43(7):995-1022. https://doi.org/10.1016/j.freeradbiomed.2007.06.026.