The 2007 NRC report on Toxicity Testing in 21st Century proposed a paradigm shift in toxicology away from animal studies focussed on apical end-points, to one using the advances in molecular omics, in vitro profiling and computational modeling to focus primarily on human relevant mechanisms and pathways.  SEAC is developing case studies including oxidative stress, to understand how the integration of in vitro data, modelling and mechanistic pathways such as those proposed within the AOP framework can inform the risk assessment process as an alternative to animal testing.  A key specific aim is to define what perturbations and at what magnitude are relevant for distinguishing adaptive from adverse effects.

Over the last few decades, extensive literature has been published on oxidative stress, a phenomenon caused by an imbalance between reactive oxygen species (ROS) production and its elimination. Indeed, oxidative stress is implicated as one of the key pathways through which many chemicals exert their toxic effects across many tissues. However due to the indispensable requirement for oxygen, cellular defenses have evolved complex adaptive mechanisms such as ROS quenching by cellular antioxidants and activation of Nrf2-KEAP1 pathway, to handle low levels of oxidative stress and maintain redox homeostasis. During oxidative stress, changes in the thiol status of the redox sensor KEAP1 result in increasing free Nrf2 which translocates into the nucleus and upregulates a range of antioxidant and Phase II metabolism-associated genes to restore oxidative balance. However, with prolonged or high levels of oxidative stress, the cellular defenses become saturated and adverse changes like protein oxidation, lipid peroxidation, and mitochondrial dysfunction become more pronounced. Defining this tipping point or ‘point of departure’ which lies at the intersection of adaptive and adverse responses, is critical for decision making within the adverse outcome pathway (AOP) framework of chemical risk assessment.

SEAC is exploring the broader network underpinning the oxidative stress pathway; identifying the molecular initiating events and mapping the cellular responses using techniques like transcriptomics, phosphoproteomics, and specific biomarker assays through high content imaging and flow cytometry to understand the changes to human relevant cell populations. A dose and time response of biomarkers for ROS production, alterations in GSH, and Nrf2 activation are being studied along with downstream effects on lipid and protein oxidation and cytotoxicity.

SEAC, and our external scientific partners are also developing a comprehensive quantitative systems biology model of the oxidative stress pathway which simulates the effect of ROS production on glutathione, protein oxidation, lipid peroxidation, mitochondrial function, and cell death via apoptosis or necrosis. Once substantiated, the model will simulate activation of adaptive feedback mechanisms like the Nrf2-KEAP1 pathway which upregulates transcription of cellular antioxidants to restore redox homeostasis at low doses of chemical.

The model, combined with biomarkers of ROS production and Nrf2 activation, can offer a useful tool to map regions of safety where cells are able to adapt and subsequently reverse functional changes, therefore enabling risk assessments within an AOP framework. The Nrf2/Keap1 pathway and its role in cellular defenses against oxidative stress, provides one of the case studies in our development and understanding of approaches to drive the TT21C vision forward with practical application.

Current collaborators – Strand Life Sciences, Hamner Institute of Life Sciences, Leiden University

Latest Presentations

Development of a mechanistic based model for Nrf2 and oxidative stress in the context of the Adverse Outcome Pathway framework

Adaptation versus Adverse Cell Responses in Oxidative Stress: Nrf2 and NFkB

Oxidative Stressors Induce Differential Cellular Responses between Human Primary and HaCaT Keratinocyte Cells

Development of an adverse outcome framework in the context of Nrf2 and oxidative stress responses to Quercetin and Curcumin

Latest Publication

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Gaurav Jain