Excessive reactive oxygen species leading to growth inhibition via uncoupling of oxidative phosphorylation and cell death

You Song^a

^a Norwegian Institute for Water Research (NIVA), Økernveien 94, NO-0579 Oslo, Norway

Reactive oxygen species (ROS) are by-products of normal cellular metabolism, but excessive ROS production can lead to oxidative stress and widespread macromolecular damage. This AOP network (AOPN) describes multiple interconnected pathways by which increased ROS can impair organismal growth. The molecular initiating event is an increase in ROS, which triggers oxidative damage to lipids, proteins, and DNA. These molecular changes converge on mitochondrial dysfunction through decreased coupling of oxidative phosphorylation and fatty acid β-oxidation, resulting in ATP depletion. In parallel, oxidative DNA damage and insufficient repair lead to DNA strand breaks and cell cycle disruption. Together, these pathways decrease cell proliferation and cell growth, promote cell injury and death, and ultimately reduce growth at the organism level. By integrating several parallel and converging biological events, this AOPN reflects the complexity of oxidative stress outcomes. It provides a mechanistic framework of high regulatory relevance, as oxidative stress is a common mode of action for many environmental contaminants. This AOPN supports the development of predictive in vitro assays, read-across strategies, and refined approaches for chemical safety and ecological risk assessment.

Reactive oxygen species (ROS) play a dual role in biology, functioning as essential signaling molecules under physiological conditions but becoming harmful when produced in excess. Imbalance between ROS generation and antioxidant defenses, commonly referred to as oxidative stress, has been linked to a wide spectrum of toxicological outcomes in both human health and environmental species. Growth impairment is of particular regulatory concern because it represents a sensitive and integrative endpoint that reflects energy status, cellular function, and organismal fitness. AOP networks (AOPNs) are especially relevant for oxidative stress, as ROS-mediated damage affects multiple cellular targets in parallel, leading to complex and interacting pathways of toxicity. Understanding these connections is critical for developing predictive toxicology tools that can account for chemical diversity and species-specific responses. The development of this AOPN is motivated by the need to capture the mechanistic complexity of oxidative stress and its consequences for growth, and to provide a transparent, structured framework that can be applied in regulatory decision-making. This AOPN is intended to support chemical risk assessment, ecological impact evaluation, and the design of alternative test methods that reduce reliance on animal testing.

The development of this AOP network (AOPN) followed a structured approach to identify, screen, and evaluate the evidence supporting the key events (KEs) and key event relationships (KERs). The strategy was designed to ensure transparency, reproducibility, and reusability of the information for regulatory applications.

The scope of the literature search was defined by the biological problem of interest: the role of reactive oxygen species (ROS) in driving oxidative stress and its downstream consequences on growth. The focus was on conserved cellular processes relevant across species (e.g., mitochondrial function, DNA damage and repair, cell proliferation, energy metabolism) and their contribution to growth impairment at the organism level.

An initial scoping review was performed to map the key domains of biology where ROS has well-established toxicological impacts. Expert input from oxidative stress and ecotoxicology research was used to prioritize candidate KEs and to identify relevant biomarkers and assays. This scoping step ensured that the AOPN captured both canonical pathways (e.g., oxidative phosphorylation) and parallel events (e.g., DNA damage responses).

A tiered literature search strategy was applied:

• Primary screening: Broad keyword searches in PubMed, Web of Science, and Scopus using combinations of terms such as reactive oxygen species, oxidative stress, mitochondrial dysfunction, oxidative phosphorylation, DNA damage, cell cycle, cell proliferation, growth impairment. Searches covered the period from 1990–2023 to include both foundational and recent advances.
• Focused surveys: Targeted searches for specific KERs, e.g., oxidative stress AND mitochondrial ATP depletion, ROS AND DNA strand breaks, oxidative stress AND growth inhibition (fish OR mammal).
• Regulatory and guidance documents: Additional screening of OECD publications, test guidelines (e.g., TG 249), and mechanistic toxicology reviews to capture regulatory relevance and assay development efforts.
• Automated tools: AOP-help tools including AOP-helpFinder and AOP-BOT were employed to systematically mine literature, extract relevant key event associations, and ensure alignment with existing AOPs in the AOP-Wiki knowledgebase. These tools provided cross-validation of manually identified KEs/KERs and highlighted potential network linkages.

Abstracts were reviewed for relevance, with exclusion of studies not addressing mechanistic links between ROS and cellular/organismal growth outcomes. Full-text evaluation was conducted for studies meeting inclusion criteria. Where available, systematic reviews and meta-analyses were prioritized.

The weight-of-evidence (WoE) approach recommended in the OECD Users’ Handbook Supplement was applied. This included evaluation of:

• Biological plausibility of each KE and KER.
• Essentiality of events, supported by experimental manipulation studies.
• Empirical support, assessed through dose-response, temporal, and incidence concordance.
• Quantitative understanding, considering thresholds and cross-species extrapolation.

The combined manual and automated strategy ensured that the AOPN is built upon a comprehensive and critically assessed evidence base. Use of tools such as AOP-helpFinder and AOP-BOT enhanced transparency, supported efficient literature mining, and facilitated re-use of individual KEs, KERs, and supporting evidence in future AOPs and AOP networks.

The biological relevance of this AOP network (AOPN) is supported by the highly conserved nature of oxidative stress mechanisms across taxa. ROS production, antioxidant defenses, mitochondrial function, and DNA damage responses are core processes in nearly all aerobic organisms, providing strong confidence in the applicability of this AOPN to both human health and ecological species. The pathway is not sex-specific, and although subtle sex-dependent differences in antioxidant capacity have been reported, the fundamental mechanisms apply broadly to males and females. Growth impairment is relevant throughout the life cycle, but early developmental stages are especially sensitive due to high energy demands and rapid cellular proliferation.

The weight of evidence (WoE) for the overall AOPN is robust. There is strong biological plausibility linking the molecular initiating event (ROS increase) to the adverse outcome (reduced growth), supported by decades of mechanistic toxicology research. Essentiality of key events is well established, as mitochondrial dysfunction, oxidative DNA damage, and disruption of cell proliferation are widely recognized as causal determinants of growth impairment. Empirical support across multiple taxa demonstrates consistent dose–response, temporal, and incidence concordance among key events. While quantitative understanding is still being refined, especially for cross-species extrapolation and thresholds of effect, the existing evidence base provides a solid foundation for application.

From a regulatory perspective, this AOPN offers value in several contexts. It can support priority setting by identifying chemicals likely to cause growth impairment through oxidative stress. It is also suitable for guiding integrated testing strategies, particularly in the development and application of in vitro assays and non-animal methods. Finally, the AOPN contributes to risk assessment by providing a mechanistic framework that links molecular perturbations to ecologically and toxicologically relevant outcomes. Overall, this AOPN provides a scientifically credible and broadly applicable basis for regulatory decision-making.

This AOP network (AOPN) has broad potential to inform regulatory decision-making, given the central role of oxidative stress in chemical toxicity and the ecological and human health relevance of growth impairment as an endpoint. Several areas of application can be envisaged:

• Test guideline development and refinement: The mechanistic framework provides a basis for designing and validating in vitro assays that capture upstream key events such as ROS production, mitochondrial dysfunction, or DNA damage. Relevant OECD Test Guidelines (e.g., TG 249: Fish Cell Line Acute Toxicity) already use cell-based measures of biomass/growth inhibition and could be refined to incorporate oxidative stress endpoints. In vitro assays developed in this context could complement or partially replace traditional apical growth studies, reducing reliance on animal testing.

• Integrated testing and assessment approaches (IATA): The AOPN can serve as a scaffold for IATA frameworks promoted by the OECD, integrating data streams from in vitro, in silico, and omics technologies. By linking early molecular events to organismal growth impairment, it supports transparent weight-of-evidence evaluations and structured regulatory decision-making.

• Chemical grouping and read-across: Because oxidative stress is a common mode of action for many structurally diverse chemicals, the AOPN can facilitate grouping and category formation. Coupled with tools such as the OECD QSAR Toolbox, chemical profilers, or structural alerts for ROS-inducing potential, the AOPN can support read-across approaches for untested substances.

• Screening-level hazard assessment: The AOPN provides a mechanistic rationale for prioritizing chemicals that induce oxidative stress for further evaluation. Screening batteries could include biomarkers of oxidative stress or intermediate key events (e.g., mitochondrial dysfunction, DNA damage) to identify potential growth-impairing agents at early stages.

• Risk assessment: By linking molecular initiating events to an ecologically and toxicologically relevant adverse outcome, the AOPN supports extrapolation from mechanistic data to population- or organism-level consequences. This mechanistic anchoring strengthens confidence in risk assessments and enhances their predictive power for both human health and environmental protection.

Overall, this AOPN contributes directly to OECD initiatives on mechanistic risk assessment, development of non-animal methods, and harmonization of predictive toxicology tools, thereby increasing its scientific and regulatory utility.