AOP ID and Title:

Short Title: ROS leading to growth inhibition via LPO and decreased cell proliferation

Authors

You Song, Li Xie, Knut Erik Tollefsen

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

 

Status

Coaches

• Shihori Tanabe

Abstract

This adverse outcome pathway (AOP 326) describes a linear route by which increased reactive oxygen species (ROS) can lead to decreased organismal growth through lipid peroxidation-mediated impairment of mitochondrial bioenergetics. 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, particularly polyunsaturated fatty acids in cellular and mitochondrial membranes, can alter membrane integrity, proton conductance, mitochondrial membrane potential, and respiratory efficiency. These effects reduce coupling of oxidative phosphorylation (OXPHOS), decrease the cellular ATP pool, impair energy-dependent cellular proliferation, and ultimately reduce organismal growth.

    AOP 326 reuses and connects established AOP-Wiki components from two important AOP contexts. The upstream oxidative stress component is associated with AOP 478, in which deposition of energy leads to oxidative stress through increased free radical generation, with subsequent oxidative molecular damage (AOP-Wiki, 2026a). This provides a curated AOP-Wiki context for the use of oxidative stress as a conserved hub KE downstream of free radical or ROS-producing stressors. The downstream bioenergetic and growth segment is directly associated with AOP 263, which causally links decreased coupling of OXPHOS to growth inhibition through ATP depletion and decreased cell proliferation and has been published in the OECD Series on Adverse Outcome Pathways (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021). Thus, AOP 326 links an oxidative-stress/lipid-damage module to an OECD-endorsed bioenergetics-to-growth module. The AOP is relevant to environmental and human health contexts because ROS production, lipid peroxidation, mitochondrial ATP generation, cell proliferation, and organismal growth are broadly conserved in aerobic eukaryotes.    Empirical support is derived from studies in algae, daphnids, mollusks, fish, mammalian systems, and human cells exposed to redox-active chemicals, metals, pesticides, hypoxia-reoxygenation, radiation, and endogenous oxidative stressors. This AOP can support mechanistic interpretation of oxidative stress-mediated growth impairment, assay selection, chemical prioritization, integrated approaches to testing and assessment (IATA), and development of quantitative AOP approaches for mitochondrial and oxidative stress-related 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.

Context

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). Lipids are important targets of oxidative attack because membrane phospholipids, especially those containing polyunsaturated fatty acyl chains, can undergo radical-driven peroxidation. Lipid peroxidation generates lipid hydroperoxides and secondary reactive products, including malondialdehyde and 4-hydroxy-2-nonenal, which can alter membrane structure, propagate oxidative damage, and affect protein and organelle function (Ayala et al., 2014).

    AOP 326 was developed to represent the lipid peroxidation-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). The downstream sequence from decreased coupling of OXPHOS to decreased ATP pool, decreased cell proliferation, and decreased growth is already represented in AOP 263 and provides the growth-relevant terminal module for AOP 326 (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021).

Strategy

AOP 326 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 purpose was to assemble a focused linear pathway from reusable AOP-Wiki elements rather than to create an isolated de novo pathway. This is particularly important because AOP 326 is one branch of the broader ROS-growth AOP network and because its KEs and KERs overlap with oxidative stress, mitochondrial dysfunction, energy metabolism, cell proliferation, and growth-related AOPs.

    Reuse of existing AOP-Wiki content was considered at the beginning of development. AOP 478 was reviewed because it provides an AOP-Wiki precedent for oxidative stress as a central KE downstream of free radical generation, and because it describes oxidative stress as a pathway by which energy deposition can damage biological molecules (AOP-Wiki, 2026a). Although AOP 478 is not a lipid peroxidation-to-growth AOP, its use of KE 1392 (Increase, Oxidative stress) and its oxidative damage context support the upstream portion of AOP 326. AOP 263 was reviewed because it provides the directly relevant downstream module consisting of KE 1446 (Decrease, Coupling of OXPHOS), KE 1771 (Decrease, ATP pool), KE 1821 (Decrease, Cell proliferation), and AO 1521 (Decrease, Growth), together with KERs 2203, 2204, and 2205 (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021). In AOP 326, the novel linking logic is therefore the connection of ROS-driven oxidative stress and lipid peroxidation to the existing OXPHOS-ATP-cell proliferation-growth module.

    The evidence base was assembled through a structured AI-human hybrid workflow. Search terms were first developed for the events in the pathway, including KE names, synonyms, endpoint names, assay terms, taxa, and species. AOP-helpFinder was then used to search PubMed for co-occurrence of KE-related terms, following approaches previously described for literature mining in support of AOP development (Carvaillo et al., 2019; Jornod et al., 2022). The exported AOP-helpFinder results included PMIDs, titles, abstracts, and matched KE terms. Overlap analysis was applied to remove redundant hits and filter literature that was unrelated to the taxa or biological systems considered relevant to this AOP.

    A large language model (LLM) was then used as an auxiliary screening and structuring tool. During abstract pre-screening, the LLM was used to extract study metadata such as stressor, species, biological system, dose or concentration, and exposure time; to identify whether a study provided evidence for biological plausibility, empirical support, essentiality, or quantitative understanding; and to flag indicators of dose-response, temporal, or incidence concordance. Studies were provisionally classified as high, medium, or low priority. High-priority studies were retrieved for full-text review, medium-priority studies were reserved for supporting evidence, and low-priority studies were documented as low relevance. For studies passing the abstract screen, the LLM was used to assist full-text review by organizing information relevant to the KER evidence table. All LLM outputs were checked manually against the full text by expert reviewers before any evidence was accepted.

    The final phase consisted of manual expert curation. Experts verified the LLM-assisted extractions, populated KER evidence tables with methods, endpoints, results, weight-of-evidence categories, and references, and made final calls for biological plausibility, empirical support, essentiality, and evidence gaps. Targeted manual searches were also used to fill gaps identified during evidence curation, especially for the lipid peroxidation to OXPHOS coupling relationship and the downstream AOP 263 KERs. Searches focused on combinations of terms such as reactive oxygen species, oxidative stress, lipid peroxidation, malondialdehyde, 4-hydroxynonenal, mitochondrial membrane potential, proton leak, oxidative phosphorylation, ATP depletion, cell proliferation, growth inhibition, paraquat, copper, cadmium, hypoxia-reoxygenation, Daphnia, algae, bivalves, fish, and AOP. Studies were prioritized when they measured two or more KEs in the same biological system, reported exposure dose or concentration and time, or provided evidence relevant to dose-response, temporal, or incidence concordance.

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Stressors

Overall Assessment of the AOP

The overall weight of evidence supporting AOP 326 is considered moderate to high. Biological plausibility is high for all six KERs in the pathway, reflecting well-established mechanistic connections between ROS, oxidative stress, lipid peroxidation, mitochondrial membrane integrity, OXPHOS coupling, ATP production, cell proliferation, and growth. The downstream bioenergetic module from decreased coupling of OXPHOS through decreased ATP pool and decreased cell proliferation to decreased growth (KERs 2203, 2204, 2205) is directly reused from OECD-endorsed AOP 263, which was published in the OECD Series on Adverse Outcome Pathways and has independently established high to moderate WoE for these relationships (OECD, 2022; Song and Villeneuve, 2021). The upstream oxidative stress-lipid peroxidation module is supported by high empirical concordance across algae, crustaceans, bivalves, fish, and mammalian systems. The novel linking relationship from lipid peroxidation to decreased OXPHOS coupling (KER 1599) is rated moderate in empirical support, as studies measuring both endpoints concurrently in the same experimental system are less common, although mechanistic evidence for cardiolipin oxidation and inner mitochondrial membrane integrity is strong. Essentiality is rated moderate to high, with the strongest support for the OXPHOS-to-ATP segment shared with AOP 263. Quantitative understanding is highest for the AOP 263-derived downstream module and low to moderate for the upstream lipid peroxidation-to-OXPHOS segment. The main uncertainties are the directionality and quantitative strength of the lipid peroxidation-to-OXPHOS relationship, given that mitochondrial dysfunction can also promote lipid peroxidation, and the potential for compensatory glycolytic ATP production to buffer depletion. AOP 326 is suitable for mechanistic interpretation, IATA development, and chemical prioritisation for oxidative stress-mediated growth impairment affecting mitochondrial energy metabolism (OECD, 2018; OECD, 2022; Becker et al., 2015).

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

The domain of applicability for AOP 326 is broad across aerobic eukaryotic organisms in which ROS generation, oxidative stress responses, lipid peroxidation, mitochondrial oxidative phosphorylation, ATP-dependent cell proliferation, and growth are biologically relevant. The AOP is most applicable to taxa and life stages in which growth depends substantially on cell proliferation and energy supply, including algae, developing aquatic organisms, juvenile fish, and proliferating mammalian or human cells. It is also relevant to adult organisms when growth, regeneration, tissue condition, or organismal size is influenced by mitochondrial energy metabolism.

The stressor domain is also broad and includes direct ROS generators, redox-cycling chemicals, metals, pesticides, mitochondrial toxicants, hypoxia-reoxygenation, and radiation. Because the MIE is defined operationally as increased ROS rather than as a chemical-specific interaction, AOP 326 should be applied to stressors that can be shown to increase ROS or oxidative stress and to produce evidence consistent with lipid peroxidation and mitochondrial bioenergetic impairment. Environmental factors such as temperature, oxygen availability, diet, lipid composition, nutrient status, and antioxidant capacity may modulate the pathway by altering ROS production, lipid susceptibility to peroxidation, mitochondrial coupling, or growth rate.

Essentiality of the Key Events

Essentiality is evaluated for the overall AOP based on whether preventing or modifying upstream KEs changes downstream KEs or the AO. Direct essentiality evidence is strongest for the AOP 263 downstream module, where removal of mitochondrial uncouplers or restoration of coupling can restore mitochondrial membrane potential and ATP production. Essentiality for lipid peroxidation is biologically plausible and supported by intervention and association studies, but direct experiments showing that blocking lipid peroxidation prevents all downstream events are less common.

 

Key event	Essentiality	Rationale	Experimental manipulation evidence (KE knock-out / inhibition / rescue)	Uncertainties
Event 1115: Reactive oxygen species, increased	Moderate	ROS scavenging or antioxidant interventions frequently attenuate oxidative stress and downstream lipid peroxidation in oxidative stress models. ROS is also used as a common early perturbation in the broader ROS-growth AOP network (Schieber and Chandel, 2014; Sies et al., 2017).	Indirect (stop/attenuation): antioxidant and ROS-scavenger pre-treatment reduces oxidative stress and downstream damage across oxidative-stress models (Schieber and Chandel, 2014; Sies et al., 2017). No selective single-source ROS knock-out is available.	ROS also participate in normal signaling; increased ROS does not always progress to adversity if antioxidant and repair systems compensate.
Event 1392: Oxidative stress, increased	Moderate to high	Oxidative stress is required for lipid peroxidation when ROS production exceeds antioxidant buffering. AOP 478 supports oxidative stress as a central KE downstream of free radical generation (AOP-Wiki, 2026a).	Indirect: modulation of antioxidant capacity alters progression to oxidative macromolecular damage; oxidative stress is the curated hub KE in endorsed AOP 478 (AOP-Wiki, 2026a; Carrothers et al., 2025).	Oxidative stress can be measured using multiple indirect biomarkers; equivalence across methods is not always clear.
Event 1445: Lipid peroxidation, increased	Moderate	Lipid peroxidation can alter membrane properties and generate reactive aldehydes that affect mitochondrial function (Ayala et al., 2014). Dietary PUFA studies in Daphnia show higher lipid peroxidation with lower mitochondrial membrane potential (Moore et al., 2023), supporting a causal role in mitochondrial impairment.	Indirect: antioxidant intervention attenuates lipid peroxidation in oxidative-stress models; direct block-and-rescue isolating LPO from other oxidative damage is uncommon (Murphy, 2009; Ouillon et al., 2021).	Direct blocking experiments are limited; lipid peroxidation may be both a cause and consequence of mitochondrial dysfunction.
Event 1446: Coupling of OXPHOS, decreased	High	This KE is reused from AOP 263. Evidence from AOP 263 supports essentiality because removal of uncouplers or restoration of coupling can recover mitochondrial membrane potential and ATP levels, reducing downstream impairment (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021).	Direct (rescue): removal of uncouplers or restoration of coupling recovers mitochondrial membrane potential and ATP in the endorsed AOP 263 module (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021).	Mild uncoupling can sometimes reduce ROS generation and may be adaptive; severity and duration determine progression.
Event 1771: ATP pool, decreased	Moderate	ATP depletion is a direct consequence of impaired OXPHOS coupling and is associated with reduced cell proliferation and cytotoxicity in multiple systems. AOP 263 identifies ATP depletion as an intermediate KE linking OXPHOS uncoupling to reduced proliferation (AOP-Wiki, 2026b; OECD, 2022).	Indirect: ATP-restoration experiments reduce downstream injury/proliferation deficits; central KE in endorsed AOP 263 (Leist et al., 1997; Nicotera et al., 1998; OECD, 2022).	Cells may compensate through glycolysis or other energy pathways; total ATP may recover transiently depending on exposure scenario.
Event 1821: Cell proliferation, decreased	Moderate	Growth depends on accumulation of cell number and biomass. AOP 263 provides evidence that decreased cell proliferation links ATP depletion to decreased growth (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021).	Indirect: proliferation deficit links bioenergetic/genotoxic upstream to growth; reused from endorsed AOP 263 with KER 2205 (AOP-Wiki, 2026d; Conlon and Raff, 1999; OECD, 2022; Song and Villeneuve, 2021).	Growth can be influenced by cell size, metabolism, development, nutrient status, and cell death, not proliferation alone.
Event 1521: Growth, decreased (AO)	Not applicable (AO)	Growth is the adverse outcome and is regulatory relevant across algae, aquatic invertebrate, fish, amphibian, and plant test systems. AOP 263 provides precedent for using decreased growth as the AO in a mitochondrial bioenergetics AOP (OECD, 2022; Song and Villeneuve, 2021).	As the adverse outcome, essentiality is assessed for upstream KEs; AOP 263 provides precedent for decreased growth as an AO downstream of these modules (OECD, 2022; Song and Villeneuve, 2021).	Growth is an integrative endpoint and can arise through multiple independent or interacting mechanisms.

 

Weight of Evidence Summary

Evidence assessment is organized by KER. Calls follow OECD weight-of-evidence considerations for biological plausibility, empirical support, and quantitative understanding (OECD, 2018, 2021).

 

Biological plausibility of KERs

KER	Biological plausibility call	Rationale
Relationship 2009: ROS increase leads to oxidative stress increase	High	The relationship is mechanistically established because oxidative stress reflects an imbalance between oxidant production and antioxidant capacity, and ROS are primary oxidant species in cellular redox biology (Schieber and Chandel, 2014; Sies et al., 2017). AOP 478 provides a curated AOP-Wiki context for oxidative stress downstream of free radical generation (AOP-Wiki, 2026a).
Relationship 3116: oxidative stress increase leads to lipid peroxidation increase	High	Free radicals and other ROS can initiate peroxidation of polyunsaturated fatty acids in membranes, generating lipid hydroperoxides and reactive aldehydes such as MDA and 4-HNE (Ayala et al., 2014; Sies et al., 2017).
Relationship 1599: lipid peroxidation increase leads to decreased coupling of OXPHOS	High	Mitochondrial coupling depends on the integrity and composition of the inner mitochondrial membrane. Lipid peroxidation can disrupt membrane properties, promote proton leak, alter membrane potential, and impair respiratory control (Murphy, 2009; Nicholls and Ferguson, 2013; Ouillon et al., 2021).
Relationship 2203: decreased coupling of OXPHOS leads to decreased ATP pool	High	This relationship is directly reused from AOP 263. OXPHOS coupling is a major determinant of ATP production in aerobic eukaryotic cells; reduced coupling lowers ATP synthesis efficiency (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021).
Relationship 2204: decreased ATP pool leads to decreased cell proliferation	High	This relationship is reused from AOP 263. Cell proliferation requires ATP for DNA replication, mitosis, biosynthesis, and maintenance of cellular processes; ATP depletion therefore plausibly reduces proliferation (AOP-Wiki, 2026b; OECD, 2022; Bonora et al., 2012; Song and Villeneuve, 2021).
Relationship 2205: decreased cell proliferation leads to decreased growth	High	This relationship is reused from AOP 263. Organismal, tissue, and population growth require accumulation of cells and biomass; sustained reduction in proliferation is therefore expected to reduce growth (AOP-Wiki, 2026b; Conlon and Raff, 1999; OECD, 2022).

 

Empirical support for KERs

KER	Empirical support call	Rationale	Inconsistencies or evidence gaps
Relationship 2009: ROS increase leads to oxidative stress increase	High	Multiple studies demonstrate concordance between ROS or ROS-producing stressors and oxidative stress markers. Paraquat increased ROS and antioxidant enzyme responses in Chlorella vulgaris (Qian et al., 2009). Paraquat also induced oxidative stress responses and lipid peroxidation in Daphnia magna (Barata et al., 2005).	ROS is often measured indirectly and may be transient; oxidative stress biomarkers vary by assay and tissue.
Relationship 3116: oxidative stress increase leads to lipid peroxidation increase	High	Oxidative stress and lipid peroxidation are often observed together. Copper increased antioxidant enzyme activity and MDA/TBARS in freshwater green microalgae (Knauert and Knauer, 2008). Paraquat and other redox-cycling stressors induced lipid peroxidation in algae and Daphnia (Barata et al., 2005; Esperanza et al., 2015; Qian et al., 2009). In the aquatic macrophyte Lemna minor, gamma radiation and the respiratory uncoupler 3,5-dichlorophenol induced a concordant oxidative stress to lipid peroxidation sequence preceding growth inhibition (Xie et al., 2018; Xie et al., 2019; Xie et al., 2022).	Lipid peroxidation can occur at different times than enzyme responses, and MDA/TBARS assays have specificity limitations.
Relationship 1599: lipid peroxidation increase leads to decreased coupling of OXPHOS	Moderate	Dietary PUFA manipulation in Daphnia showed higher lipid peroxidation associated with lower mitochondrial membrane potential (Moore et al., 2023). Cyclic hypoxia in Mya arenaria increased proton leak and reduced OXPHOS coupling efficiency, consistent with oxidative lipid and membrane damage effects on mitochondrial coupling (Ouillon et al., 2021). In Lemna minor, gamma radiation and 3,5-dichlorophenol reduced mitochondrial membrane potential downstream of lipid peroxidation, providing primary-producer support for the lipid-peroxidation to OXPHOS-coupling link (Xie et al., 2018; Xie et al., 2019).	Direct studies measuring lipid peroxidation and OXPHOS coupling in the same exposure series are limited; mitochondrial dysfunction can also promote lipid peroxidation, complicating directionality.
Relationship 2203: decreased coupling of OXPHOS leads to decreased ATP pool	High	AOP 263 reports strong evidence for this KER. Experimental studies with mitochondrial toxicants and uncouplers show concordance between impaired mitochondrial function and reduced ATP production across cell types and taxa (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021). Cadmium exposure in oysters reduced state 3 respiration and affected mitochondrial bioenergetics (Sokolova et al., 2005).	Compensatory glycolysis or altered metabolic demand can obscure total ATP changes.
Relationship 2204: decreased ATP pool leads to decreased cell proliferation	Moderate to high	AOP 263 provides curated evidence that ATP depletion is associated with reduced cell proliferation, with dose and incidence concordance in several systems (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021). In Chlamydomonas reinhardtii, paraquat caused ATP depletion and cell injury/death or growth-related effects in multiple-endpoint assays (Nestler et al., 2012; Jamers and De Coen, 2010).	Total ATP assays may partly reflect cell number or viability; separating ATP depletion from cytotoxicity requires careful study design.
Relationship 2205: decreased cell proliferation leads to decreased growth	Moderate	AOP 263 supports this KER using evidence that reduced proliferation contributes to growth inhibition across taxa (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021). Algal cell density and growth rate endpoints are directly linked to cell proliferation; reduced proliferation also supports developmental growth impairment in animals.	Growth is influenced by both cell number and cell size, as well as energy allocation, development, and environmental conditions.

Inconsistencies and uncertainties

The main uncertainty for AOP 326 is the directionality and quantitative strength of the lipid peroxidation to OXPHOS coupling relationship. Lipid peroxidation can impair mitochondrial membranes, but mitochondrial dysfunction can also enhance ROS generation and thereby increase lipid peroxidation. The linear AOP represents one biologically plausible and empirically supported direction within a broader feedback-prone network. A second uncertainty is that mild mitochondrial uncoupling may reduce ROS production and serve as an adaptive response, whereas severe or sustained uncoupling reduces ATP synthesis and supports adverse outcomes. Finally, growth is a multifactorial endpoint; reduced cell proliferation is an important contributor, but organismal growth may also be influenced by nutrient status, development, endocrine regulation, cell death, and environmental factors.

Quantitative Consideration

Quantitative understanding varies across the AOP. The downstream AOP 263 module has the strongest quantitative foundation, whereas upstream oxidative stress and lipid peroxidation relationships are more often described qualitatively or semi-quantitatively.

 

KER	Quantitative understanding call	Rationale
2009: ROS increase to oxidative stress increase	Low to moderate	ROS measurements are reactive, transient, and assay-dependent. Quantitative relationships can be developed within a defined assay system, but generalizable prediction across taxa and stressors remains limited (Sies et al., 2017).
3116: oxidative stress increase to lipid peroxidation increase	Low to moderate	Dose-response relationships are reported for oxidative stress markers and lipid peroxidation in several systems, but assay differences and lipid composition strongly affect response magnitude (Ayala et al., 2014; Knauert and Knauer, 2008).
1599: lipid peroxidation increase to decreased OXPHOS coupling	Low to moderate	Some quantitative associations exist between lipid peroxidation and mitochondrial membrane potential or coupling efficiency, but models generalizable across taxa and membrane types are not yet established (Moore et al., 2023; Ouillon et al., 2021).
2203: decreased OXPHOS coupling to decreased ATP pool	High	AOP 263 reports strong quantitative understanding, supported by bioenergetic theory and models linking mitochondrial coupling and ATP production (AOP-Wiki, 2026b; OECD, 2022; Song and Villeneuve, 2021).
2204: decreased ATP pool to decreased cell proliferation	Moderate	Quantitative relationships are available in defined cell systems, but thresholds depend on cell type, metabolic state, and viability effects (AOP-Wiki, 2026b; OECD, 2022).
2205: decreased cell proliferation to decreased growth	Moderate	Growth models provide quantitative relationships between proliferation and tissue or organismal growth, but extrapolation across species and exposure contexts remains uncertain (Conlon and Raff, 1999; OECD, 2022).

 

BMD/POD-anchored concordance

The following benchmark-dose/point-of-departure (BMD/POD) concordance table anchors AOP 326 to quantitative cross-KE ordering, in line with Handbook section 4C. The multiomics point-of-departure (moPOD) dataset for gamma-irradiated Daphnia magna (Song et al., 2023) provides POD magnitudes for increased ROS, decreased ATP, decreased OXPHOS coupling, and cell death, demonstrating the expected upstream-to-downstream POD ordering (more sensitive PODs upstream). The moPOD is presented as POD magnitude evidence, not as a causal re-ordering of KEs. The Lemna minor EDR50 range provides a whole-pathway apical anchor in an aquatic primary producer.

 

Key event (functional category)	POD metric	POD value (mGy/h)	POD ordering	Source
KE 1115: ROS, increased (mROS)	moPOD (multiomics POD)	0.4	1 (most sensitive)	Song et al., 2023
KE 1771: ATP pool, decreased	moPOD	2.5	2	Song et al., 2023
KE 1446: OXPHOS coupling, decreased (UPS/OXPHOS module)	moPOD	42.3	3	Song et al., 2023
KE 55: Cell injury/death (apoptosis)	moPOD	42.3	3 (least sensitive)	Song et al., 2023
Upstream KE chain → growth (Lemna minor, gamma)	EDR50 (growth)	31.5–54.8 (mGy/h)	whole-pathway apical	Xie et al., 2018, 2019, 2022

 

Considerations for Potential Applications of the AOP (optional)

AOP 326 can support mechanistic interpretation of growth impairment caused by oxidative stressors that induce lipid peroxidation and mitochondrial bioenergetic dysfunction. 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, OXPHOS impairment, ATP depletion, reduced proliferation, 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, and proliferation assays, to an apical growth endpoint.

AOP 326 can also support chemical grouping and read-across for stressors that share evidence of oxidative lipid damage and mitochondrial bioenergetic impairment. 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 used as part of a weight-of-evidence framework that considers stressor mode of action, exposure context, taxonomic relevance, assay specificity, and concordance across multiple KEs. The AOP also highlights important method-development needs, particularly standardized assays for lipid peroxidation, mitochondrial coupling, and ATP depletion that can be applied across taxa and integrated into quantitative AOP approaches.

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Sokolova IM, Sokolov EP, Ponnappa KM. 2005. Cadmium exposure affects mitochondrial bioenergetics and gene expression of key mitochondrial proteins in the eastern oyster Crassostrea virginica Gmelin (Bivalvia: Ostreidae). Aquatic Toxicology 73:242-255.

Song Y, Villeneuve DL. 2021. Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased cell proliferation. Environmental Toxicology and Chemistry 40:2951-2963.

Xie L, Gomes T, Solhaug KA, Song Y, Tollefsen KE. 2018. Linking mode of action of the model respiratory and photosynthesis uncoupler 3,5-dichlorophenol to adverse outcomes in Lemna minor. Aquatic Toxicology 197:98-108. https://doi.org/10.1016/j.aquatox.2018.02.005

Xie L, Solhaug KA, Song Y, Brede DA, Lind OC, Salbu B, Tollefsen KE. 2019. Modes of action and adverse effects of gamma radiation in an aquatic macrophyte Lemna minor. Science of the Total Environment 680:23-34. https://doi.org/10.1016/j.scitotenv.2019.05.016

Xie L, Song Y, Petersen K, Solhaug KA, Lind OC, Brede DA, Salbu B, Tollefsen KE. 2022. Ultraviolet B modulates gamma radiation-induced stress responses in Lemna minor at multiple levels of biological organisation. Science of the Total Environment 846:157457. https://doi.org/10.1016/j.scitotenv.2022.157457

Appendix 1

List of MIEs in this AOP

Event: 1115: Increase, Reactive oxygen species

Short Name: Increase, ROS

Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

ROS is a normal constituent found in all organisms, lifestages, and sexes.

Key Event Description

Biological State: increased reactive oxygen species (ROS)

Biological compartment: an entire cell -- may be cytosolic, may also enter organelles.

Reactive oxygen species (ROS) are O₂- derived molecules that can be both free radicals (e.g. superoxide, hydroxyl, peroxyl, alcoxyl) and non-radicals (hypochlorous acid, ozone and singlet oxygen) (Bedard and Krause 2007; Ozcan and Ogun 2015). ROS production occurs naturally in all kinds of tissues inside various cellular compartments, such as mitochondria and peroxisomes (Drew and Leeuwenburgh 2002; Ozcan and Ogun 2015). Furthermore, these molecules have an important function in the regulation of several biological processes – they might act as antimicrobial agents or triggers of animal gamete activation and capacitation (Goud et al. 2008; Parrish 2010; Bisht et al. 2017). 
However, in environmental stress situations (exposure to radiation, chemicals, high temperatures) these molecules have its levels drastically increased, and overly interact with macromolecules, namely nucleic acids, proteins, carbohydrates and lipids, causing cell and tissue damage (Brieger et al. 2012; Ozcan and Ogun 2015). 

How it is Measured or Detected

Photocolorimetric assays (Sharma et al. 2017; Griendling et al. 2016) or through commercial kits purchased from specialized companies.

Yuan, Yan, et al., (2013) described ROS monitoring by using H₂-DCF-DA, a redox-sensitive fluorescent dye. Briefly, the harvested cells were incubated with H₂-DCF-DA (50 µmol/L final concentration) for 30 min in the dark at 37°C. After treatment, cells were immediately washed twice, re-suspended in PBS, and analyzed on a BD-FACS Aria flow cytometry. ROS generation was based on fluorescent intensity which was recorded by excitation at 504 nm and emission at 529 nm.

Lipid peroxidation (LPO) can be measured as an indicator of oxidative stress damage Yen, Cheng Chien, et al., (2013).

Chattopadhyay, Sukumar, et al. (2002) assayed the generation of free radicals within the cells and their extracellular release in the medium by addition of yellow NBT salt solution (Park et al., 1968). Extracellular release of ROS converted NBT to a purple colored formazan. The cells were incubated with 100 ml of 1 mg/ml NBT solution for 1 h at 37 °C and the product formed was assayed at 550 nm in an Anthos 2001 plate reader. The observations of the ‘cell-free system’ were confirmed by cytological examination of parallel set of explants stained with chromogenic reactions for NO and ROS.

On the basis of the pathogenesis of drug-induced phototoxicity, a reactive oxygen species (ROS) assay was proposed to evaluate the phototoxic risk of chemicals. The ROS assay can monitor generation of ROS, such as singlet oxygen and superoxide, from photoirradiated chemicals, and the ROS data can be used to evaluate the photoreactivity of chemicals (Onoue et al. , 2014, Onoue et al. , 2013, Onoue and Tsuda, 2006).  The ROS assay is a recommended approach by guidelines to evaluate the phototoxic risk of chemicals (ICH, 2014, PCPC, 2014).

References

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Granger, D. N. and Kvietys, P. R. (2015). "Reperfusion injury and reactive oxygen species: The evolution of a concept" Redox Biol. doi: 10.1016/j.redox.2015.08.020. PMID: 26484802; PMCID: PMC4625011.

Griendling, K. K., et al. (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): e39-75.

Griendling, Kathy K., Rhian M. Touyz, Jay L. Zweier, Sergey Dikalov, William Chilian, Yeong-Renn Chen, David G. Harrison, Aruni Bhatnagar, and American Heart Association Council on Basic Cardiovascular Sciences. 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): e39–75.

ICH. ICH Guideline S10 Guidance on Photosafety Evaluation of Pharmaceuticals.: International Council on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use; 2014.

Itziou, A., et al. (2011). "In vivo and in vitro effects of metals in reactive oxygen species production, protein carbonylation, and DNA damage in land snails Eobania vermiculata." Archives of Environmental Contamination and Toxicology, 60(4), 697–707. https://doi.org/10.1007/s00244-010-9583-5

Ji, W. O., et al. "Quantitation of the ROS production in plasma and radiation treatments of biotargets." Sci Rep. 2019 Dec 27;9(1):19837. doi: 10.1038/s41598-019-56160-0. PMID: 31882663; PMCID: PMC6934759.

Kruk, J. and Aboul-Enein, H. Y. (2017). "Reactive Oxygen and Nitrogen Species in Carcinogenesis: Implications of Oxidative Stress on the Progression and Development of Several Cancer Types." Mini-Reviews in Medicinal Chemistry, 17:11. doi:10.2174/1389557517666170228115324

Lee, D. Y., et al. (2020). "PEGylated Bilirubin-coated Iron Oxide Nanoparticles as a Biosensor for Magnetic Relaxation Switching-based ROS Detection in Whole Blood." Theranostics, 10(5), 1997-2007. doi:10.7150/thno.39662

Li, Z., et al. (2020). "Inhibition of MiR-25 attenuates doxorubicin-induced apoptosis, reactive oxygen species production and DNA damage by targeting pten." International Journal of Medical Sciences, 17(10), 1415–1427. https://doi.org/10.7150/ijms.41980

Liou, G. Y. and Storz, P. "Reactive oxygen species in cancer." Free Radic Res. 2010 May;44(5):479-96. doi:10.3109/10715761003667554. PMID: 20370557; PMCID: PMC3880197.

Lu, Y., et al. (2010). "Phosphatidylinositol-3-kinase/akt regulates bleomycin-induced fibroblast proliferation and collagen production." American journal of respiratory cell and molecular biology, 42(4), 432–441. https://doi.org/10.1165/rcmb.2009-0002OC

Onoue, S., et al. (2013). "Establishment and intra-/inter-laboratory validation of a standard protocol of reactive oxygen species assay for chemical photosafety evaluation." J Appl Toxicol. 33(11):1241-50. doi: 10.1002/jat.2776. Epub 2012 Jun 13. PMID: 22696462.

Onoue S, Hosoi K, Toda T, Takagi H, Osaki N, Matsumoto Y, et al. Intra-/inter-laboratory validation study on reactive oxygen species assay for chemical photosafety evaluation using two different solar simulators. Toxicology in vitro : an international journal published in association with BIBRA. 2014;28:515-23.

Onoue S, Hosoi K, Wakuri S, Iwase Y, Yamamoto T, Matsuoka N, et al. Establishment and intra-/inter-laboratory validation of a standard protocol of reactive oxygen species assay for chemical photosafety evaluation. Journal of applied toxicology : JAT. 2013;33:1241-50.

Onoue S, Kawamura K, Igarashi N, Zhou Y, Fujikawa M, Yamada H, et al. Reactive oxygen species assay-based risk assessment of drug-induced phototoxicity: classification criteria and application to drug candidates. J Pharm Biomed Anal. 2008;47:967-72.

Onoue S, Seto Y, Gandy G, Yamada S. Drug-induced phototoxicity; an early in vitro identification of phototoxic potential of new drug entities in drug discovery and development. Current drug safety. 2009;4:123-36.

Onoue S, Tsuda Y. Analytical studies on the prediction of photosensitive/phototoxic potential of pharmaceutical substances. Pharmaceutical research. 2006;23:156-64.

Ozcan, Ayla, and Metin Ogun. 2015. “Biochemistry of Reactive Oxygen and Nitrogen Species.” In Basic Principles and Clinical Significance of Oxidative Stress, edited by Sivakumar Joghi Thatha Gowder. Rijeka: IntechOpen.

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PCPC. PCPC 2014 safety evaluation guidelines; Chapter 7: Evaluation of Photoirritation and Photoallergy potential. Personal Care Products Council; 2014.

Ramos, M. F. P., et al. (2018). "Xanthine oxidase inhibitors and sepsis." Int J Immunopathol Pharmacol. 32:2058738418772210. doi:10.1177/2058738418772210

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Sharma, Gunjan, Nishant Kumar Rana, Priya Singh, Pradeep Dubey, Daya Shankar Pandey, and Biplob Koch. 2017. “p53 Dependent Apoptosis and Cell Cycle Delay Induced by Heteroleptic Complexes in Human Cervical Cancer Cells.” Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie 88 (April): 218–31.

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Zhang, Z., et al. (2011). "Reactive oxygen species mediate arsenic induced cell transformation and tumorigenesis through Wnt/β-catenin pathway in human colorectal adenocarcinoma DLD1 cells. " Toxicology and Applied Pharmacology, 256(2), 114-121. doi:10.1016/j.taap.2011.07.016

List of Key Events in the AOP

Event: 1392: Increase, Oxidative Stress

Short Name: Increase, Oxidative Stress

Event Component

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic applicability: Occurrence of oxidative stress is not species specific.  

Life stage applicability: Occurrence of oxidative stress is not life stage specific. 

Sex applicability: Occurrence of oxidative stress is not sex specific. 

Evidence for perturbation by prototypic stressor: There is evidence of the increase of oxidative stress following perturbation from a variety of stressors including exposure to ionizing radiation and altered gravity (Bai et al., 2020; Ungvari et al., 2013; Zhang et al., 2009).  

Key Event Description

Oxidative stress is defined as an imbalance in the production of reactive oxygen species (ROS) and antioxidant defenses. High levels of oxidizing free radicals can be very damaging to cells and molecules within the cell.  As a result, the cell has important defense mechanisms to protect itself from ROS. For example, Nrf2 is a transcription factor and master regulator of the oxidative stress response. During periods of oxidative stress, Nrf2-dependent changes in gene expression are important in regaining cellular homeostasis (Nguyen, et al., 2009) and can be used as indicators of the presence of oxidative stress in the cell. 

In addition to the directly damaging actions of ROS, cellular oxidative stress also changes cellular activities on a molecular level. Redox sensitive proteins have altered physiology in the presence and absence of ROS, which is caused by the oxidation of sulfhydryls to disulfides on neighboring amino acids (Antelmann & Helmann 2011). Importantly Keap1, the negative regulator of Nrf2, is regulated in this manner (Itoh, et al. 2010). 

ROS also undermine the mitochondrial defense system from oxidative damage. The antioxidant systems consist of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, as well as antioxidants such as α-tocopherol and ubiquinol, or antioxidant vitamins and minerals including vitamin E, C, carotene, lutein, zeaxanthin, selenium, and zinc (Fletcher, 2010). The enzymes, vitamins and minerals catalyze the conversion of ROS to non-toxic molecules such as water and O2. However, these antioxidant systems are not perfect and endogenous metabolic processes and/or exogenous oxidative influences can trigger cumulative oxidative injuries to the mitochondria, causing a decline in their functionality and efficiency, which further promotes cellular oxidative stress (Balasubramanian, 2000; Ganea & Harding, 2006; Guo et al., 2013; Karimi et al., 2017).  

However, an emerging viewpoint suggests that ROS-induced modifications may not be as detrimental as previously thought, but rather contribute to signaling processes (Foyer et al., 2017). 

 

Sources of ROS Production 

Direct Sources: Direct sources involve the deposition of energy onto water molecules, breaking them into active radical species. When ionizing radiation hits water, it breaks it into hydrogen (H*) and hydroxyl (OH*) radicals by destroying its bonds. The hydrogen will create hydroxyperoxyl free radicals (HO2*) if oxygen is available, which can then react with another of itself to form hydrogen peroxide (H2O2) and more O2 (Elgazzar and Kazem, 2015). Antioxidant mechanisms are also affected by radiation, with catalase (CAT) and peroxidase (POD) levels rising as a result of exposure (Seen et al. 2018; Ahmad et al. 2021).  

Indirect Sources: An indirect source of ROS is the mitochondria, which is one of the primary producers in eukaryotic cells (Powers et al., 2008).  As much as 2% of the electrons that should be going through the electron transport chain in the mitochondria escape, allowing them an opportunity to interact with surrounding structures. Electron-oxygen reactions result in free radical production, including the formation of hydrogen peroxide (H2O2) (Zhao et al., 2019). The electron transport chain, which also creates ROS, is activated by free adenosine diphosphate (ADP), O2, and inorganic phosphate (Pi) (Hargreaves et al. 2020; Raimondi et al. 2020; Vargas-Mendoza et al. 2021). The first and third complexes of the transport chain are the most relevant to mammalian ROS production (Raimondi et al., 2020). The mitochondria has its own set of DNA and it is a prime target of oxidative damage (Guo et al., 2013). ROS is also produced through nicotinamide adenine dinucleotide phosphate oxidase (Nox) stimulation, an event commenced by angiotensin II, a product/effector of the renin-angiotensin system (Nguyen Dinh Cat et al. 2013; Forrester et al. 2018). Other ROS producers include xanthine oxidase, immune cells (macrophage, neutrophils, monocytes, and eosinophils), phospholipase A2 (PLA2), monoamine oxidase (MAO), and carbon-based nanomaterials (Powers et al. 2008; Jacobsen et al. 2008; Vargas-Mendoza et al. 2021). 

How it is Measured or Detected

Oxidative Stress: Direct measurement of ROS is difficult because ROS are unstable. The presence of ROS can be assayed indirectly by measurement of cellular antioxidants, or by ROS-dependent cellular damage. Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed 

• Detection of ROS by chemiluminescence (https://www.sciencedirect.com/science/article/abs/pii/S0165993606001683) 
• Detection of ROS by chemiluminescence is also described in OECD TG 495 to assess phototoxic potential. 
• Glutathione (GSH) depletion. GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green- ab138881.html). 
• TBARS. Oxidative damage to lipids can be measured by assaying for lipid peroxidation using TBARS (thiobarbituric acid reactive substances) using a commercially available kit. 
• 8-oxo-dG. Oxidative damage to nucleic acids can be assayed by measuring 8-oxo-dG adducts (for which there are a number of ELISA based commercially available kits),or HPLC, described in Chepelev et al. (Chepelev, et al. 2015). 

  

Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assay for Nrf2 activity include: 

• Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus Western blot for increased Nrf2 protein levels 
• Western blot of cytoplasmic and nuclear fractions to observe translocation of Nrf2 protein from the cytoplasm to the nucleus qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences) 
• Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway (e.g., Jackson et al. 2014) 
• OECD TG422D describes an ARE-Nrf2 Luciferase test method 

In general, there are a variety of commercially available colorimetric or fluorescent kits for detecting Nrf2 activation.

Assay Type & Measured Content	Description	Dose Range Studied	Assay Characteristics (Length/Ease of use/Accuracy)
ROS  Formation in the Mitochondria assay (Shaki et al., 2012)	“The mitochondrial ROS measurement was performed flow cytometry using DCFH-DA. Briefly, isolated kidney mitochondria were incubated with UA (0, 50, 100 and 200 µM) in respiration buffer containing (0.32 mM sucrose, 10mM Tris, 20 mM Mops, 50 µM EGTA, 0.5 mM MgCl2, 0.1 mM KH2PO4 and 5 mM sodium succinate) [32]. In the interval times of 5, 30 and 60 min following the UA addition, a sample was taken and DCFH-DA was added (final concentration, 10 µM) to mitochondria and was then incubated for 10 min.Uranyl acetate-induced ROS generation in isolated kidney mitochondria were determined through the flow cytometry (Partec, Deutschland) equipped with a 488-nm argon ion laser and supplied with the Flomax software and the signals were obtained using a 530-nm bandpass filter (FL-1 channel). Each determination is based on the mean fluorescence intensity of 15,000 counts.”	0, 50,100 and 200 µM of Uranyl Acetate	Long/ Easy High accuracy
Mitochondrial Antioxidant Content Assay Measuring GSH content (Shaki et al., 2012)	“GSH content was determined using DTNB as the indicator and spectrophotometer method for the isolated mitochondria. The mitochondrial fractions (0.5 mg protein/ml) were incubated with various concentrations of uranyl acetate for 1 h at 30 °C and then 0.1 ml of mitochondrial fractions was added into 0.1 mol/l of phosphate buffers and 0.04% DTNB in a total volume of 3.0 ml (pH 7.4). The developed yellow color was read at 412 nm on a spectrophotometer (UV-1601 PC, Shimadzu, Japan). GSH content was expressed as µg/mg protein.”	0, 50,  100, or  200 µM  Uranyl Acetate
H2O2 Production Assay Measuring H2O2 Production in isolated mitochondria (Heyno et al., 2008)	“Effect of CdCl2 and antimycin A (AA) on H2O2 production in isolated mitochondria from potato. H2O2 production was measured as scopoletin oxidation. Mitochondria were incubated for 30 min in the measuring buffer  (see the Materials and Methods) containing 0.5 mM succinate as an electron donor and 0.2 µM mesoxalonitrile 3‐chlorophenylhydrazone (CCCP) as an uncoupler, 10 U horseradish peroxidase and 5 µM scopoletin.”	0, 10, 30  µM Cd2+     2 µM antimycin A
Flow Cytometry ROS & Cell Viability (Kruiderig et al., 1997)	“For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At t 5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)”“For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At t 5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)”		Strong/easy medium
DCFH-DA  Assay Detection of hydrogen peroxide production (Yuan et al.,  2016)	Intracellular ROS production was measured using DCFH-DA as a probe. Hydrogen peroxide oxidizes DCFH to DCF. The probe is hydrolyzed intracellularly to DCFH carboxylate anion. No direct reaction with H2O2 to form fluorescent production.	0-400  µM	Long/ Easy High accuracy
H2-DCF-DAAssay Detection of superoxide production (Thiebault etal., 2007)	This dye is a stable nonpolar compound which diffuses readily into the cells and yields H2-DCF. Intracellular OH or ONOO- react with H2-DCF when cells contain peroxides, to form the highly fluorescent compound DCF, which effluxes the cell. Fluorescence intensity of DCF is measured using a fluorescence spectrophotometer.	0–600  µM	Long/ Easy High accuracy
CM-H2DCFDA  Assay (Eruslanov  & Kusmartsev, 2009)	The dye (CM-H2DCFDA) diffuses into the cell and is cleaved by esterases, the thiol reactive chlormethyl group reacts with intracellular glutathione which can be detected using flow cytometry.		Long/Easy/ High Accuracy

 

Method of Measurement	References	Description	OECD-Approved Assay
Chemiluminescence	(Lu, C. et al., 2006;   Griendling, K. K., et al., 2016)	ROS can induce electron transitions in molecules, leading to electronically excited products. When the electrons transition back to ground state, chemiluminescence is emitted and can be measured. Reagents such as luminol and lucigenin are commonly used to amplify the signal.	No
Spectrophotometry	(Griendling, K. K., et al., 2016)	NO has a short half-life. However, if it has been reduced to nitrite (NO2-), stable azocompounds can be formed via the Griess Reaction, and further measured by spectrophotometry.	No
Direct or Spin Trapping-Based electron paramagnetic resonance (EPR) Spectroscopy	(Griendling, K. K., et al., 2016)	The unpaired electrons (free radicals) found in ROS can be detected with EPR and is known as electron paramagnetic resonance. A variety of spin traps can be used.	No
Nitroblue Tetrazolium Assay	(Griendling, K. K., et al., 2016)	The Nitroblue Tetrazolium assay is used to measure O2.− levels. O2.− reduces nitroblue tetrazolium (a yellow dye) to formazan (a blue dye), and can be measured at 620 nm.	No
Fluorescence analysis of dihydroethidium (DHE) or Hydrocyans	(Griendling, K. K., et al., 2016)	Fluorescence analysis of DHE is used to measure O2.− levels.  O2.− is reduced to O2 as DHE is oxidized to 2-hydroxyethidium, and this reaction can be measured by fluorescence. Similarly, hydrocyans can be oxidized by any ROS, and measured via fluorescence.	No
Amplex Red Assay	(Griendling, K. K., et al., 2016)	Fluorescence analysis to measure extramitochondrial or extracellular H2O2 levels. In the presence of horseradish peroxidase and H2O2, Amplex Red is oxidized to resorufin, a fluorescent molecule measurable by plate reader.	No
Dichlorodihydrofluorescein Diacetate (DCFH-DA)	(Griendling, K. K., et al., 2016)	An indirect fluorescence analysis to measure intracellular H2O2 levels.  H2O2 interacts with peroxidase or heme proteins, which further react with DCFH, oxidizing it to dichlorofluorescein (DCF), a fluorescent product.	No
HyPer Probe	(Griendling, K. K., et al., 2016)	Fluorescent measurement of intracellular H2O2 levels. HyPer is a genetically encoded fluorescent sensor that can be used for in vivo and in situ imaging.	No
Cytochrome c Reduction Assay	(Griendling, K. K., et al., 2016)	The cytochrome c reduction assay is used to measure O2.− levels. O O2.− is reduced to O2 as ferricytochrome c is oxidized to ferrocytochrome c, and this reaction can be measured by an absorbance increase at 550 nm.	No
Proton-electron double-resonance imaging (PEDRI)	(Griendling, K. K., et al., 2016)	The redox state of tissue is detected through nuclear magnetic resonance/magnetic resonance imaging, with the use of a nitroxide spin probe or biradical molecule.	No
Glutathione (GSH) depletion	(Biesemann, N. et al., 2018)	A downstream target of the Nrf2 pathway is involved in GSH synthesis. As an indication of oxidation status, GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html).	No
Thiobarbituric acid reactive substances (TBARS)	(Griendling, K. K., et al., 2016)	Oxidative damage to lipids can be measured by assaying for lipid peroxidation with TBARS using a commercially available kit.	No
Protein oxidation (carbonylation)	(Azimzadeh et al., 2017; Azimzadeh et al., 2015; Ping et al., 2020)	Can be determined with ELISA or a commercial assay kit. Protein oxidation can indicate the level of oxidative stress.	No
Seahorse XFp Analyzer	Leung et al. 2018	The Seahorse XFp Analyzer provides information on mitochondrial function, oxidative stress, and metabolic dysfunction of viable cells by measuring respiration (oxygen consumption rate; OCR) and extracellular pH (extracellular acidification rate; ECAR).	No

 

Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assays for Nrf2 activity include:  

Method of Measurement	References	Description	OECD-Approved Assay
Immunohistochemistry	(Amsen, D., de Visser, K. E., and Town, T., 2009)	Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus	No
qPCR	(Forlenza et al., 2012)	qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences)	No
Whole transcriptome profiling via microarray or via RNA-seq followed by a pathway analysis	(Jackson, A. F. et al., 2014)	Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway	No

 

References

Ahmad, S. et al. (2021), “60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression”, Journal Insect Science, Vol. 21/5, Oxford University Press, Oxford, https://doi.org/10.1093/jisesa/ieab080 

Antelmann, H. and J. D. Helmann (2011), “Thiol-based redox switches and gene regulation.”, Antioxidants & Redox Signaling, Vol. 14/6, Mary Ann Leibert Inc., Larchmont, https://doi.org/10.1089/ars.2010.3400 

Amsen, D., de Visser, K. E., and Town, T. (2009), “Approaches to determine expression of inflammatory cytokines”, in Inflammation and Cancer, Humana Press, Totowa, https://doi.org/10.1007/978-1-59745-447-6_5  

Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction”, Journal of Proteome Research, Vol. 14/2, American Chemical Society, Washington, https://doi.org/10.1021/pr501141b 

Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, International Journal of Radiation Biology, Vol. 93/9, Informa, London, https://doi.org/10.1080/09553002.2017.1339332 

Azzam, E. I. et al. (2012), “Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury”, Cancer Letters, Vol. 327/1-2, Elsevier, Ireland, https://doi.org/10.1016/j.canlet.2011.12.012 

Bai, J. et al. (2020), “Irradiation-induced senescence of bone marrow mesenchymal stem cells aggravates osteogenic differentiation dysfunction via paracrine signaling”, American Journal of Physiology - Cell Physiology, Vol. 318/5, American Physiological Society, Rockville, https://doi.org/10.1152/ajpcell.00520.2019. 

Balasubramanian, D (2000), “Ultraviolet radiation and cataract”, Journal of ocular pharmacology and therapeutics, Vol. 16/3, Mary Ann Liebert Inc., Larchmont, https://doi.org/10.1089/jop.2000.16.285.   

Biesemann, N. et al., (2018), “High Throughput Screening of Mitochondrial Bioenergetics in Human Differentiated Myotubes Identifies Novel Enhancers of Muscle Performance in Aged Mice”, Scientific Reports, Vol. 8/1, Nature Portfolio, London, https://doi.org/10.1038/s41598-018-27614-8.  

Elgazzar, A. and N. Kazem. (2015), “Chapter 23: Biological effects of ionizing radiation” in The Pathophysiologic Basis of Nuclear Medicine, Springer, New York, pp. 540-548 

Eruslanov, E., & Kusmartsev, S. (2010). Identification of ROS using oxidized DCFDA and flow-cytometry. Methods in molecular biology ,N.J.,  Vol. 594,  https://doi.org/10.1007/978-1-60761-411-1_4 

Fletcher, A. E (2010), “Free radicals, antioxidants and eye diseases: evidence from epidemiological studies on cataract and age-related macular degeneration”, Ophthalmic Research, Vol. 44, Karger International, Basel, https://doi.org/10.1159/000316476.  

Forlenza, M. et al. (2012), “The use of real-time quantitative PCR for the analysis of cytokine mRNA levels” in Cytokine Protocols, Springer, New York, https://doi.org/10.1007/978-1-61779-439-1_2  

Forrester, S.J. et al. (2018), “Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology”, Physiological Reviews, Vol. 98/3, American Physiological Society, Rockville, https://doi.org/10.1152/physrev.00038.201 

Foyer, C. H., A. V. Ruban, and G. Noctor (2017), “Viewing oxidative stress through the lens of oxidative signalling rather than damage”, Biochemical Journal, Vol. 474/6, Portland Press, England, https://doi.org/10.1042/BCJ20160814 

Ganea, E. and J. J. Harding (2006), “Glutathione-related enzymes and the eye”, Current eye research, Vol. 31/1, Informa, London, https://doi.org/10.1080/02713680500477347.  

Griendling, K. K. et al. (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, Vol. 119/5, Lippincott Williams & Wilkins, Philadelphia, https://doi.org/10.1161/RES.0000000000000110  

Guo, C. et al. (2013), “Oxidative stress, mitochondrial damage and neurodegenerative diseases”, Neural regeneration research, Vol. 8/21, Publishing House of Neural Regeneration Research, China, https://doi.org/10.3969/j.issn.1673-5374.2013.21.009 

Hargreaves, M., and L. L. Spriet (2020), “Skeletal muscle energy metabolism during exercise.”, Nature Metabolism, Vol. 2, Nature Portfolio, London, https://doi.org/10.1038/s42255-020-0251-4 

Hladik, D. and S. Tapio (2016), “Effects of ionizing radiation on the mammalian brain”, Mutation Research/Reviews in Mutation Research, Vol. 770, Elsevier, Amsterdam, https://doi.org/10.1016/j.mrrev.2016.08.003 

Itoh, K., J. Mimura and M. Yamamoto (2010), “Discovery of the negative regulator of Nrf2, Keap1: a historical overview”, Antioxidants & Redox Signaling, Vol. 13/11, Mary Ann Leibert Inc., Larchmont, https://doi.org/10.1089/ars.2010.3222  

Jackson, A.F. et al. (2014), “Case study on the utility of hepatic global gene expression profiling in the risk assessment of the carcinogen furan.”, Toxicology and Applied Pharmacology, Vol. 274/11, Elsevier, Amsterdam, https://doi.org/10.1016/j.taap.2013.10.019 

Jacobsen, N.R. et al. (2008), “Genotoxicity, cytotoxicity, and reactive oxygen species induced by single-walled carbon nanotubes and C60 fullerenes in the FE1-MutaTM Mouse lung epithelial cells”, Environmental and Molecular Mutagenesis, Vol. 49/6, John Wiley & Sons, Inc., Hoboken, https://doi.org/10.1002/em.20406 

Karimi, N. et al. (2017), “Radioprotective effect of hesperidin on reducing oxidative stress in the lens tissue of rats”, International Journal of Pharmaceutical Investigation, Vol. 7/3, Phcog Net, Bengaluru, https://doi.org/10.4103/jphi.JPHI_60_17.  

Leung, D.T.H., and Chu, S. (2018), “Measurement of Oxidative Stress: Mitochondrial Function Using the Seahorse System” In: Murthi, P., Vaillancourt, C. (eds) Preeclampsia. Methods in Molecular Biology, vol 1710. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7498-6_22 

Lu, C., G. Song, and J. Lin (2006), “Reactive oxygen species and their chemiluminescence-detection methods”, TrAC Trends in Analytical Chemistry, Vol. 25/10, Elsevier, Amsterdam, https://doi.org/10.1016/j.trac.2006.07.007 

Nguyen Dinh Cat, A. et al. (2013), “Angiotensin II, NADPH oxidase, and redox signaling in the vasculature”, Antioxidants & redox signaling, Vol. 19/10, Mary Ann Liebert, Larchmont, https://doi.org/10.1089/ars.2012.4641 

Ping, Z. et al. (2020), “Oxidative Stress in Radiation-Induced Cardiotoxicity”, Oxidative Medicine and Cellular Longevity, Vol. 2020, Hindawi, https://doi.org/10.1155/2020/3579143 

Powers, S.K. and M.J. Jackson. (2008), “Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production”, Physiological Reviews, Vol. 88/4, American Physiological Society, Rockville, https://doi.org/10.1152/physrev.00031.2007 

Raimondi, V., F. Ciccarese and V. Ciminale. (2020), “Oncogenic pathways and the electron transport chain: a dangeROS liason”, British Journal of Cancer, Vol. 122/2, Nature Portfolio, London, https://doi.org/10.1038/s41416-019-0651-y 

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Vargas-Mendoza, N. et al. (2021), “Oxidative Stress, Mitochondrial Function and Adaptation to Exercise: New Perspectives in Nutrition”, Life, Vol. 11/11, Multidisciplinary Digital Publishing Institute, Basel, https://doi.org/10.3390/life11111269 

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Event: 1445: Increase, Lipid peroxidation

Short Name: Increase, LPO

Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

The biological domain of applicability for this KE is broad because lipid membranes and oxidizable fatty acids are widely conserved biological features. The event is applicable wherever lipid substrates susceptible to oxidation are present and where oxidants can access those substrates. The KE is therefore relevant across many biological systems, including unicellular algae, invertebrates, fish, mammals and human-derived cells. The current evidence base is strongest in mammalian systems because lipid peroxidation chemistry and analytical methods have been extensively studied there, but ecotoxicological evidence supports relevance in algae, crustaceans, mollusks and fish.

    The KE is not intrinsically limited by sex or life stage. However, the magnitude of lipid peroxidation and its downstream consequences may be modified by lipid composition, antioxidant capacity, oxygen availability, temperature, metabolic rate, nutritional status, metal availability, and exposure duration. Organisms or tissues enriched in polyunsaturated fatty acids, exposed to high oxygen flux, or experiencing antioxidant depletion may be particularly susceptible. In photosynthetic organisms, lipid peroxidation may also occur in chloroplast and thylakoid membranes; in animals, mitochondria and plasma membranes are common sites of interest.

    Within the ROS-growth AOP network, this KE is especially relevant as a molecular damage event linking oxidative stress to impaired mitochondrial membrane function, decreased coupling of oxidative phosphorylation, reduced ATP production, cell injury, and decreased growth. Nevertheless, this KE should remain modular: it may be reused in other AOPs whenever increased lipid oxidation products are measured as a consequence of oxidative stress or other lipid-damaging perturbations.

 

Key Event Description

Lipid peroxidation is an oxidative degradation process affecting lipids, particularly polyunsaturated fatty acids in cellular and organelle membranes. The process is initiated when oxidants, including free radicals and reactive oxygen species, abstract hydrogen atoms from susceptible lipid chains. This generates lipid radicals that react with molecular oxygen to form lipid peroxyl radicals and lipid hydroperoxides. These products can propagate chain reactions, producing additional oxidized lipids and secondary reactive aldehydes such as malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE), and related hydroxyalkenals (Esterbauer et al., 1991; Yin et al., 2011; Ayala et al., 2014).

    As a key event, increased lipid peroxidation represents a measurable increase in oxidized lipid products relative to an appropriate control state. The event may reflect direct oxidative damage to membrane lipids, increased formation of lipid hydroperoxides, increased accumulation of MDA or 4-HNE, or increased abundance of specific oxidized phospholipid or fatty acid species. Because lipid peroxidation products can alter membrane fluidity, permeability and signaling, the event is relevant both as a marker of oxidative damage and as a potential contributor to downstream mitochondrial dysfunction, loss of membrane integrity, cytotoxicity and impaired growth (Esterbauer et al., 1991; Uchida, 2003; Ayala et al., 2014).

This KE should be described independently of any specific upstream or downstream event. In an AOP context, lipid peroxidation is commonly downstream of oxidative stress and upstream of events related to decreased mitochondrial coupling, cellular injury, or altered membrane-dependent biological processes. However, the KE itself is defined only by the increased lipid oxidation state and its measurable biochemical products.

How it is Measured or Detected

No OECD Test Guideline is currently dedicated specifically to measurement of lipid peroxidation as a standalone endpoint. Nevertheless, the KE can be measured using several well-established biochemical and analytical methods. Scientific confidence is highest when methods quantify specific lipid peroxidation products or oxidized lipid species directly, and lower when nonspecific colorimetric assays are used without appropriate controls or confirmatory methods.

 

Measurement approach	Endpoint measured	Representative method names	Scientific confidence and limitations
TBARS / MDA assays	Thiobarbituric acid reactive substances, often interpreted as MDA or MDA-like products	TBARS assay; spectrophotometric or fluorometric MDA assays	Widely used and sensitive, but not fully specific because TBA can react with compounds other than MDA. Best used as a screening or comparative indicator of lipid peroxidation, particularly when supported by extraction, HPLC separation or additional markers (Buege and Aust, 1978; Ohkawa et al., 1979; Janero, 1990; Draper and Hadley, 1990).
4-HNE and hydroxyalkenal assays	4-hydroxy-2-nonenal and related reactive aldehydes	ELISA, immunoblotting of HNE-protein adducts, HPLC or LC-MS quantification	Mechanistically informative because 4-HNE is a major bioactive lipid peroxidation product. Antibody-based methods can detect protein adducts, whereas chromatographic or mass spectrometric methods improve specificity (Esterbauer et al., 1991; Uchida, 2003; Ayala et al., 2014).
Lipid hydroperoxide assays	Primary lipid hydroperoxides	FOX assay; iodometric assays; commercial lipid hydroperoxide kits	Useful for detecting relatively early lipid peroxidation products. Hydroperoxides can be unstable and sample handling is critical. FOX-based methods provide a simple approach for lipid hydroperoxide detection (Jiang et al., 1992).
Chromatography and mass spectrometry	Specific oxidized fatty acids, oxidized phospholipids, oxylipins or oxidized lipid classes	HPLC, GC, LC-MS/MS, lipidomics	High specificity and quantitative power when standards and validated workflows are available. These methods can distinguish individual oxidized lipid species and are preferred for detailed mechanistic studies (Yin et al., 2011; Li et al., 2019).
Fluorescent probes and imaging	Oxidation-sensitive fluorescent signal in cellular lipids	BODIPY 581/591 C11 and related lipid oxidation probes	Useful for cell-based or imaging applications and spatial localization, but probe specificity, photoxidation and calibration must be considered. Best used with complementary biochemical or analytical endpoints.

 

 

References

AOP-Wiki. 2026. Key Event 1445: Increase, Lipid peroxidation. AOP-Wiki. Available at: https://aopwiki.org/events/1445. Accessed 14 May 2026.

Alam MR, Ehiguese FO, Vitale D, Martín-Díaz ML. 2022. Oxidative stress response to hydrogen peroxide exposure of Mytilus galloprovincialis and Ruditapes philippinarum: reduced embryogenesis success and altered biochemical response of sentinel marine bivalve species. Environmental Chemistry and Ecotoxicology 4:97-105.

Ayala A, Munoz MF, Arguelles S. 2014. Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine and Cellular Longevity 2014:360438. https://doi.org/10.1155/2014/360438.

Belaid C, Sbartai I. 2021. Assessing the effects of Thiram to oxidative stress responses in a freshwater bioindicator cladoceran (Daphnia magna). Chemosphere 268:128808. https://doi.org/10.1016/j.chemosphere.2020.128808.

Buege JA, Aust SD. 1978. Microsomal lipid peroxidation. Methods in Enzymology 52:302-310. https://doi.org/10.1016/S0076-6879(78)52032-6.

Cong B, Liu C, Wang L, Chai Y. 2020. The impact on antioxidant enzyme activity and related gene expression following adult zebrafish (Danio rerio) exposure to dimethyl phthalate. Animals 10(4):717. https://doi.org/10.3390/ani10040717.

Draper HH, Hadley M. 1990. Malondialdehyde determination as index of lipid peroxidation. Methods in Enzymology 186:421-431. https://doi.org/10.1016/0076-6879(90)86135-I.

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.

Esterbauer H, Schaur RJ, Zollner H. 1991. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radical Biology and Medicine 11(1):81-128. https://doi.org/10.1016/0891-5849(91)90192-6.

Janero DR. 1990. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radical Biology and Medicine 9(6):515-540. https://doi.org/10.1016/0891-5849(90)90131-2.

Jiang ZY, Hunt JV, Wolff SP. 1992. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Analytical Biochemistry 202(2):384-389. https://doi.org/10.1016/0003-2697(92)90122-N.

Knauert S, Knauer K. 2008. The role of reactive oxygen species in copper toxicity to two freshwater green algae. Journal of Phycology 44(2):311-319. https://doi.org/10.1111/j.1529-8817.2008.00471.x.

Li L, Zhong S, Shen X, Li Q, Xu W, Tao Y, Yin H. 2019. Recent development on liquid chromatography-mass spectrometry analysis of oxidized lipids. Free Radical Biology and Medicine 144:16-34. https://doi.org/10.1016/j.freeradbiomed.2019.06.006.

Moore TD, Martin-Creuzburg D, Yampolsky LY. 2023. Diet effects on longevity, heat tolerance, lipid peroxidation and mitochondrial membrane potential in Daphnia. Oecologia 202(1):151-163. https://doi.org/10.1007/s00442-023-05382-1.

Ohkawa H, Ohishi N, Yagi K. 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95(2):351-358. https://doi.org/10.1016/0003-2697(79)90738-3.

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.

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.

Uchida K. 2003. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Progress in Lipid Research 42(4):318-343. https://doi.org/10.1016/S0163-7827(03)00014-6.

Yin H, Xu L, Porter NA. 2011. Free radical lipid peroxidation: mechanisms and analysis. Chemical Reviews 111(10):5944-5972. https://doi.org/10.1021/cr200084z.

Event: 1446: Decrease, Coupling of oxidative phosphorylation

Short Name: Decrease, Coupling of OXPHOS

Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic applicability domain

This key event is in general considered applicable to most eukaryotes, as the mitochondrion and oxidative phosphorylation are highly conserved (Roger 2017).

 

Life stage applicability domain

This key event is considered applicable to all life stages, as ATP synthesis by oxidative phosphorylation is an essential biological process for most living organisms.

 

Sex applicability domain

This key event is considered sex-unspecific, as both males and females use oxidative phosphorylation as a main process to generate ATP.

Key Event Description

Decreased coupling of oxidative phosphorylation (OXPHOS), or uncoupling of OXPHOS, describes dissipation of protonmotive force (PMF) across the inner mitochondrial membrane (IMM) by environmental stressors. In eukaryotes, the mitochondrial electron transport chain mediates a series of redox reactions to create a PMF across the IMM. The PMF is used as energy to drive adenosine triphosphate (ATP) synthesis through phosphorylation of adenosine diphosphate (ADP). These processes are coupled and referred to as OXPHOS. A number of chemicals can dissipate the PMF, leading to uncoupling of OXPHOS. This key event describes the main outcome of the interactions between an uncoupler and the transmembrane PMF. An uncoupler can bind to a proton in the mitochondrial inter membrane space, transport the proton to the matrix side of the IMM, release the proton and move back to the inter membrane space. These processes are repeated until the transmembrane PMF is dissipated. This KE is therefore a lumped term of these processes and represents the final consequence of the interactions.

How it is Measured or Detected

Uncoupling of oxidative phosphorylation can be indicated by reduced mitochondrial membrane potential, increased proton leak and/or increased oxygen consumption rate.

• Mitochondrial membrane potential can be determined using ToxCast high-throughput screening bioassays such as “APR_HepG2_MitoMembPot”, “APR_Hepat_MitoFxnI”, and “APR_Mitochondrial_membrane_potential”, and the Tox21 high-throughput screening assay “tox21-mitotox-p1”.
• Mitochondrial membrane potential can also be measured using commercially available fluorescent probes such as TMRM (tetramethylrhodamine, methyl ester, perchlorate), TMRE (tetramethylrhodamine, ethyl ester, perchlorate) and JC-1 (Perry 2011).
• Proton leak and oxygen consumption rate can be measured using a high-resolution respirometry (Affourtit 2018) or a Seahorse XF analyzer (Divakaruni 2014).

References

Affourtit C, Wong H-S, Brand MD. 2018. Measurement of proton leak in isolated mitochondria. In Palmeira CM, Moreno AJ, eds, Mitochondrial Bioenergetics: Methods and Protocols. Springer New York, New York, NY, pp 157-170.

Attene-Ramos MS, Huang R, Sakamuru S, Witt KL, Beeson GC, Shou L, Schnellmann RG, Beeson CC, Tice RR, Austin CP, Xia M. 2013. Systematic study of mitochondrial toxicity of environmental chemicals using quantitative high throughput screening. Chemical Research in Toxicology 26:1323-1332. DOI: 10.1021/tx4001754.

Attene-Ramos MS, Huang RL, Michael S, Witt KL, Richard A, Tice RR, Simeonov A, Austin CP, Xia MH. 2015. Profiling of the Tox21 chemical collection for mitochondrial function to identify compounds that acutely decrease mitochondrial membrane potential. Environ Health Persp 123:49-56. DOI: 10.1289/ehp.1408642.

Divakaruni AS, Paradyse A, Ferrick DA, Murphy AN, Jastroch M. 2014. Chapter Sixteen - Analysis and Interpretation of Microplate-Based Oxygen Consumption and pH Data. In Murphy AN, Chan DC, eds, Methods in Enzymology. Vol 547. Academic Press, pp 309-354.

Dreier DA, Denslow ND, Martyniuk CJ. 2019. Computational in vitro toxicology uncovers chemical structures impairing mitochondrial membrane potential. J Chem Inf Model 59:702-712. DOI: 10.1021/acs.jcim.8b00433.

Escher BI, Schwarzenbach RP. 2002. Mechanistic studies on baseline toxicity and uncoupling of organic compounds as a basis for modeling effective membrane concentrations in aquatic organisms. Aquatic Sciences 64:20-35. DOI: 10.1007/s00027-002-8052-2.

Legradi J, Dahlberg A-K, Cenijn P, Marsh G, Asplund L, Bergman Å, Legler J. 2014. Disruption of Oxidative Phosphorylation (OXPHOS) by Hydroxylated Polybrominated Diphenyl Ethers (OH-PBDEs) Present in the Marine Environment. Environmental Science & Technology 48:14703-14711. DOI: 10.1021/es5039744.

Naven RT, Swiss R, Klug-Mcleod J, Will Y, Greene N. 2012. The development of structure-activity relationships for mitochondrial dysfunction: Uncoupling of oxidative phosphorylation. Toxicol Sci 131:271-278. DOI: 10.1093/toxsci/kfs279.

Perry SW, Norman JP, Barbieri J, Brown EB, Gelbard HA. 2011. Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. BioTechniques 50:98-115. DOI: 10.2144/000113610.

Roger AJ, Munoz-Gomez SA, Kamikawa R. 2017. The origin and diversification of mitochondria. Curr Biol 27:R1177-R1192. DOI: 10.1016/j.cub.2017.09.015.

Russom CL, Bradbury SP, Broderius SJ, Hammermeister DE, Drummond RA. 1997. Predicting modes of toxic action from chemical structure: Acute toxicity in the fathead minnow (Pimephales promelas). Environ Toxicol Chem 16:948-967. DOI: https://doi.org/10.1002/etc.5620160514.

Schultz TW, Cronin MTD. 1997. Quantitative structure-activity relationships for weak acid respiratory uncouplers to Vibrio fisheri. Environ Toxicol Chem 16:357-360. DOI: https://doi.org/10.1002/etc.5620160235.

Shim J, Weatherly LM, Luc RH, Dorman MT, Neilson A, Ng R, Kim CH, Millard PJ, Gosse JA. 2016. Triclosan is a mitochondrial uncoupler in live zebrafish. J Appl Toxicol 36:1662-1667. DOI: 10.1002/jat.3311.

Sugiyama Y, Shudo T, Hosokawa S, Watanabe A, Nakano M, Kakizuka A. 2019. Emodin, as a mitochondrial uncoupler, induces strong decreases in adenosine triphosphate (ATP) levels and proliferation of B16F10 cells, owing to their poor glycolytic reserve. Genes to Cells 24:569-584. DOI: https://doi.org/10.1111/gtc.12712.

Terada H. 1990. Uncouplers of oxidative phosphorylation. Environ Health Perspect 87:213-218. DOI: 10.1289/ehp.9087213.

Troger F, Delp J, Funke M, van der Stel W, Colas C, Leist M, van de Water B, Ecker GF. 2020. Identification of mitochondrial toxicants by combined in silico and in vitro studies – A structure-based view on the adverse outcome pathway. Computational Toxicology 14:100123. DOI: https://doi.org/10.1016/j.comtox.2020.100123.

Weatherly LM, Shim J, Hashmi HN, Kennedy RH, Hess ST, Gosse JA. 2016. Antimicrobial agent triclosan is a proton ionophore uncoupler of mitochondria in living rat and human mast cells and in primary human keratinocytes. Journal of Applied Toxicology 36:777-789. DOI: https://doi.org/10.1002/jat.3209.

Xia M, Huang R, Shi Q, Boyd WA, Zhao J, Sun N, Rice JR, Dunlap PE, Hackstadt AJ, Bridge MF, Smith MV, Dai S, Zheng W, Chu PH, Gerhold D, Witt KL, DeVito M, Freedman JH, Austin CP, Houck KA, Thomas RS, Paules RS, Tice RR, Simeonov A. 2018. Comprehensive analyses and prioritization of Tox21 10K chemicals affecting mitochondrial function by in-depth mechanistic studies. Environ Health Perspect 126:077010. DOI: 10.1289/EHP2589.

Event: 1771: Decrease, Adenosine triphosphate pool

Short Name: Decrease, ATP pool

Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic applicability domain

This key event is in general considered applicable to all eukaryotes utilizing ATP as a direct source of energy and signaling molecule.

 

Life stage applicability domain

This key event is considered applicable to all life stages, as all developmental stages require energy supply to maintain necessary physiological processes.

 

Sex applicability domain

This key event is considered sex-unspecific, as both males and females use ATP as an essential energy molecule.

Key Event Description

Decreased adenosine triphosphate (ATP) pool describes the loss of balance between ATP synthesis and ATP consumption, leading to reduced total ATP. As a primary form of biological energy, ATP is used by many biological processes (Bonora 2012). Decrease in ATP level normally attributes to metabolic disorders in major ATP synthetic pathways, such as mitochondrial oxidative phosphorylation, fatty acid β-oxidation, glycolysis and plant photophosphorylation.

How it is Measured or Detected

-The ATP pool in cells or tissue can be quantified using a well-established ATP bioluminescent assay (Lemasters 1978; Wibom 1990). Assay principles: ATP can react with luciferase and luciferin from firefly and the luminescence emitted from the reaction is proportional to the ATP concentration:

ATP + D-Luciferin + O₂ è Oxyluciferin + AMP + PPi + CO₂ + Light

-ToxCast high-throughput screening bioassays, such as “NCCT_HEK293T_CellTiterGLO” and “NIS_HEK293T_CTG_Cytotoxicity” can be used to measure this KE.

 

References

Bonora M, Patergnani S, Rimessi A, De Marchi E, Suski JM, Bononi A, Giorgi C, Marchi S, Missiroli S, Poletti F, Wieckowski MR, Pinton P. 2012. ATP synthesis and storage. Purinergic Signalling 8:343-357. DOI: 10.1007/s11302-012-9305-8.

Lemasters JJ, Hackenbrock CR. 1978. [4] Firefly luciferase assay for ATP production by mitochondria. Methods in Enzymology. Vol 57. Academic Press, pp 36-50.

Wibom R, Lundin A, Hultman E. 1990. A sensitive method for measuring ATP-formation in rat muscle mitochondria. Scandinavian Journal of Clinical and Laboratory Investigation 50:143-152. DOI: 10.1080/00365519009089146.

Event: 1821: Decrease, Cell proliferation

Short Name: Decrease, Cell proliferation

Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic applicability domain

This key event is in general applicable to all eukaryotes, as most organisms are known to use cell proliferation to achieve growth.

 

Life stage applicability domain

This key event is in general applicable to all life stages. As cell proliferation not only occurs in developing organisms, but also in adults.

 

Sex applicability domain

This key event is sex-unspecific, as both genders use the same cell proliferation mechanisms.

Key Event Description

Decreased cell proliferation describes the outcome of reduced cell division and cell growth. Cell proliferation is considered the main mechanism of tissue and organismal growth (Conlon 1999). Decreased cell proliferation has been associated with abnormal growth-factor signaling and cellular energy depletion (DeBerardinis 2008).

How it is Measured or Detected

Multiple types of in vitro bioassays can be used to measure this key event:

• ToxCast high-throughput screening bioassays such as “BSK_3C_Proliferation”, “BSK_CASM3C_Proliferation” and “BSK_SAg_Proliferation” can be used to measure cell proliferation status.
• Commercially available methods such as the well-established 5-bromo-2’-deoxyuridine (BrdU) (Raza 1985; Muir 1990) or 5-ethynyl-2’-deoxyuridine (EdU) assay. Both assays measure DNA synthesis in dividing cells to indicate proliferation status.

References

Conlon I, Raff M. 1999. Size control in animal development. Cell 96:235-244. DOI: 10.1016/s0092-8674(00)80563-2.

DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. 2008. The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metabolism 7:11-20. DOI: https://doi.org/10.1016/j.cmet.2007.10.002.

Muir D, Varon S, Manthorpe M. 1990. An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures. Analytical Biochemistry 185:377-382. DOI: https://doi.org/10.1016/0003-2697(90)90310-6.

Raza A, Spiridonidis C, Ucar K, Mayers G, Bankert R, Preisler HD. 1985. Double labeling of S-phase murine cells with bromodeoxyuridine and a second DNA-specific probe. Cancer Research 45:2283-2287.

List of Adverse Outcomes in this AOP

Event: 1521: Decrease, Growth

Short Name: Decrease, Growth

Event Component

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic applicability domain

This key event is in general applicable to all eukaryotes.

 

Life stage applicability domain

This key event is applicable to early life stages such as embryo and juvenile.

 

Sex applicability domain

This key event is sex-unspecific.

Key Event Description

Decreased growth refers to a reduction in size and/or weight of a tissue, organ or individual organism. Growth is normally controlled by growth factors and mainly achieved through cell proliferation (Conlon 1999).

How it is Measured or Detected

Growth can be indicated by measuring weight, length, total volume, and/or total area of a tissue, organ or individual organism.  

Regulatory Significance of the AO

Growth is a regulatory relevant chronic toxicity endpoint for almost all organisms. Multiple OECD test guidelines have included growth either as a main endpoint of concern, or as an additional endpoint to be considered in the toxicity assessments. Relevant test guidelines include, but not only limited to:

 

-Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test

-Test No. 208: Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test

-Test No. 211: Daphnia magna Reproduction Test

-Test No. 212: Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages

-Test No. 215: Fish, Juvenile Growth Test

-Test No. 221: Lemna sp. Growth Inhibition Test

-Test No. 228: Determination of Developmental Toxicity to Dipteran Dung Flies (Scathophaga stercoraria L. (Scathophagidae), Musca autumnalis De Geer (Muscidae))

-Test No. 241: The Larval Amphibian Growth and Development Assay (LAGDA)

-Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents

-Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents

-Test No. 416: Two-Generation Reproduction Toxicity

-Test No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

-Test No. 443: Extended One-Generation Reproductive Toxicity Study

-Test No. 453: Combined Chronic Toxicity/Carcinogenicity Studies

References

Conlon I, Raff M. 1999. Size control in animal development. Cell 96:235-244. DOI: 10.1016/s0092-8674(00)80563-2.

Appendix 2

List of Key Event Relationships in the AOP