A Closer Look at Oxidative Stress


I had the opportunity to serve as an SOT Reporter for the Symposium entitled “Role of Oxidative Stress in Health and Disease: Mechanisms, Methods of Detection, and Biomarkers” just at the end of SOT 58th Annual Meeting and ToxExpo at the Baltimore Convention Center in Baltimore, Maryland, on Wednesday, March 13, 2019. I am a doctoral degree candidate in environmental toxicology at Texas Tech University, and I was particularly interested in this Symposium to expand my knowledge in this area of my research interest. The Symposium was chaired by Dr. Bhagavatula Moorthy of Baylor College of Medicine in Houston, Texas, and co-chaired by Dr. Lynette Rogers of the Research Institute at Nationwide Children’s Hospital in Columbus, Ohio.

Oxidative stress may occur when there is an imbalance of redox homeostasis between oxidants and antioxidants favoring the oxidants such as reactive oxygen species (ROS), leading to disruption of redox signaling and subsequently causing cellular injury and impairment of many cellular functions. There are several studies on toxic exposures that result in oxidative stress, many of which seemed to provide a mechanistic exposure-outcome (cause-effect or stressor-response) relationship. This Symposium, therefore, focused on the mechanisms of toxicity of a wide variety of chemicals that mediate toxicity via oxidative stress. The Symposium was endorsed by the Molecular and Systems Biology Specialty Section and the Mechanisms Specialty Section.

The overall goal of the session was to discuss the molecular and cellular mechanisms by which ROS contribute to oxidative stress and alter various signaling pathways, inducing toxicities in target organs and causing human neurodegenerative and pulmonary diseases as well as cancer.  

The specific goals were to present discussions of (1) methods of detection and measurement of oxidative stress, including integrated metabolomics; (2) the role of epigenetic mechanisms in the developing lung; (3) the mechanistic role of cytochrome P450 (CYP) enzymes and Nrf2 in oxidative stress; and (4) novel biomarkers to distinguish between oxidative stress and inflammation.

The chemistry of ROS, the uttrasensitive methods to detect and analyze them in vivo, mechanisms by which they contribute to organ toxicities, and translational research leading to the development of rational strategies (e.g., discovery of novel drugs in collaboration with the pharmaceutical industry) for the prevention and/or treatment of human diseases caused by oxidative stress were discussed.

There were five presentations covered during the Symposium: (1) “ROS-Mediated Epigenetic Changes in Developing Lungs”; (2) “NRF2, Oxidative Stress, and Inflammatory Lung Injury”; (3) “Validation of Best Detection Methods for Oxidized Macromolecules In Vivo and in Smokers”; (4) “Redox Metabolism and Oxidative Stress”; and (5) “Mechanistic Role of Cytochrome P4501A and 1B1 Enzymes in the Metabolism of Reactive Oxygen Species (ROS)-Mediated Formation of Lipid Hydroperoxides: Implications for Hyperoxic Lung Injury and Human ARDS.” At the end of each presentation, questions were taken from the audience.

The first presentation, “ROS-Mediated Epigenetic Changes in Developing Lungs,” was given by Dr. Lynette Rogers of the Research Institute at Nationwide Children’s Hospital in Columbus, Ohio. This presentation covered three main points, including the following: (1) epigenetic modifications, including DNA methylation, histone modification, and microRNAs; (2) oxygen toxicity and ROS; and (3) a model of newborn hyperoxia and methylation of DNA and histones. Also, Dr. Rogers talked about adult consequences of being born extremely preterm, which include impairment of lung function, requiring long-term respiratory care. An important takeaway from this presentation was “The Oxygen Paradox,” which states the necessity of oxygen for survival of all aerobic cells while it is also a universal cell poison, which may lead to ROS generation that results in epigenetic changes.

In the study by Dr. Rogers and her group, they found out that:

  • Epigenetic modifications linked to adult disease can be initiated by an adverse perinatal environment.
  • Perinatal inflammation results in epigenetic changes (e.g., increased DNA methylation, decreased histone methylation).
  • ROS-induced increase in methyltransferase activity results in greater methylation of CpG islands and histone proteins.

This presentation offered data that are current on methylation changes to DNA and histone proteins.

The second talk, “NRF2, Oxidative Stress, and Inflammatory Lung Injury,” was presented by Dr. Donna Zhang of the University of Arizona in Tucson, Arizona. This presentation covered the following: (1) discovery of the antioxidant responsive elements (AREs) in phase II genes of detoxification enzymes and antioxidants, (2) activation of ARE-dependent genes by transcription factor NRF2 via NRF2-sMAF dimerization and subsequent binding to AREs initiate transcription, (3) Nrf2 regulation by Keap1 and Nrf2 targeted genes in cellular stress response, and (4) non-muscle myosin light chain kinase isoform (nmMLCK) in endothelial barrier integrity.

In the study by Dr. Zhang and her group, the following were identified:

  • A novel NRF2-mediated repression of the MYLK gene, which encodes nmMLCK.
  • Replication protein A1 (RPA1) as a novel binding partner of NRF2 with a competitive advantage over NRF2-sMAF to bind ARE.
  • NRF2-RPA1-ARE-NNRS complex suppresses the expression of genes involved in various cellular processes (e.g., tumor suppression).
  • NRF2-mediated suppression of target genes (e.g., MYLK) in human lung endothelium and preclinical lung injury mouse models.

The findings in the study by Dr. Zhang and her group revealed a paradigm shift in the mechanism of NRF2 function in the activation and repression of transcription. Therefore, the study presents the potential to target NRF2-mediated suppression in treating lung pathologies.

Dr. Maria Kadiiska of the National Institute of Environmental Health Sciences (NIEHS) in Research Triangle Park, North Carolina, gave the third presentation, “Validation of Best Detection Methods for Oxidized Macromolecules In Vivo and in Smokers.” This presentation covered the following: (1) mechanistic differences between inflammation and oxidative stress; (2) diseases associated with oxidative stress/inflammation (e.g., cancer); (3) significance of oxidative stress and why its measurement is not used clinically; (4) NIEHS objective of biomarkers of oxidative stress study (BOSS) as noninvasive biomarker, consistent, accurate, sensitive, specific, and selective; (5) the need for different models of oxidative stress (e.g., varying organ responses, organ-specific oxidative stress versus systemic stress); and (6) interpretation of inflammation and oxidative stress using F2-isoprostanes. They tested three dose groups (control, low, and high) in three animal models [chloroform (CCl4), ozone (O3), lipopolysaccharide (LPS)] temporally at five time points. Afterward, they measured in blood, plasma, and urine: antioxidants (GSH/GSSG, AsA, vit. C, etc.), oxidation products of lipid (e.g., TBARs, MDA, F2-isoprostanes), protein (e.g., carbonyls), and DNA (strand breaks, 8-OH-dG, etc.), using different techniques (e.g., HPLC, GC/MS, ELISA, UV/Vis).

In the study by Dr. Kadiiska and her group, they identified the following:

  • Biomarkers that are measurable indicators of oxidative stress include GSH/GSSG, MDA, AsA, and F2-isoprostanes.
  • Biomarkers that are not indicators of oxidative stress include protein carbonyls, TBARs, 8-OH-dG, strand break (comet assay), and vit. C.
  • Time- and dose-dependent increase in plasma and urinary concentrations of MDA and F2-isoprostanes in LPS-treated minipigs.
  • F2-isoprostanes as the best marker of oxidative stress in plasma and urine of CCl4 and LPS.
  • F2-isoprostanes may be generated via both free radical and enzymatic pathways and as such may serve as a biomarker of both inflammation (predominant) and oxidative stress.
  • Establishment of a method to simultaneously assess both inflammation and oxidative stress in both an LPS animal model and humans (smokers and nonsmokers).

Dr. Kadiiska concluded that measuring the 8-iso-PGF2α / PGF2α ratio should be considered in lieu of measuring increased 8-iso-PGF2α alone to distinguish between oxidative stress and inflammation in vitro, in vivo, and in humans. This finding calls for re-examination and new interpretation of literature studies that have identified and interpreted oxidative stress as increased 8-iso-PGF2α alone.

The fourth presentation, “Redox Metabolism and Oxidative Stress,” was given by Dr. Dean Jones of Emory University in Atlanta, Georgia. This presentation covered the following: (1) the current definition of oxidative stress and position for redefinition in terms of disruption of thiol redox circuits; (2) principles of redox regulation in biological systems organization (The Redox Code); (3) the redox metabolome and proteome in providing an adaptive network system to respond to environmental challenges; (4) systems biology: transition from Cartesian reductionist to holistic systems approaches (bottom-up and top-down development of mitochondrial systems); (5) xMWAS, a software to integrate multiple ‘omics datasets with exposures and outcomes data; and (6) the redox interface (exposome, proteome, epigenome, genome) and redox effects of non-redox active metal, cadmium (Cd).

Studies by Dr. Jones and his group identified the following:

  • Very low oral Cd effect on mouse response to H1N1 infection.
  • Low-dose Cd increases inflammatory lung pathology.
  • Liver mitochondrial proteins are oxidized following Cd exposure.
  • Acute Cd exposure inactivates glutaredoxin, inhibits glutathionyl disulfides reduction, and induces apoptosis.
  • The software xMWAS enables the understanding of complex toxic responses.

As described by Dr. Jones, multiple systems function together as a network to protect against oxidative stress. Since most biologic responses to toxicants are either adaptive or protective mechanisms, xMWAS (utilizing redox organization/regulation principles, redox indicators, ‘omics technologies, and big data analysis) can be used to distinguish between both mechanistic responses. Therefore, in toxicology research on oxidative stress, integrated redox systems approaches are suitable.

Dr. Bhagavatula Moorthy of Baylor College of Medicine in Houston, Texas, presented the fifth talk, “Mechanistic Role of Cytochrome P4501A and 1B1 Enzymes in the Metabolism of Reactive Oxygen Species (ROS)-Mediated Formation of Lipid Hydroperoxides: Implications for Hyperoxic Lung Injury and Human ARDS.” This presentation covered the following: (1) hyperoxia and lung injury with reference to bronchopulmonary dysplasia (BPD), (2) hyperoxia and cytochrome P450 (CYP) enzymes, (3) hyperoxia and pulmonary FICZ levels, and (4) hyperoxia and hepatic oxidative DNA lesions/adducts. Hypotheses of their study include that hyperoxia induces CYP1A by Ah receptor–dependent mechanisms, mice lacking Cyp1a2 or 1b1 will show altered susceptibility to hyperoxic lung injury, and lipid peroxidation products and oxidative DNA adducts will increase in livers and lungs of hyperoxia-exposed mice.

Dr. Moorthy and his group observed the following in their study:

  • Accelerated death of CYP1A2 (-/-) mice in hyperoxia.
  • Increased susceptibility of CYP1A1- and CYP1A2-null mice to hyperoxic lung injury coupled with increased levels of pulmonary F2-isoprostanes and isofurans.
  • CYP1B1-null mice showed lesser susceptibility to oxygen injury.
  • Oxygen-induced formation of bulky oxidative lesions/adducts in CYP1A2-null mice.

From the results of the study by Dr. Moorthy and his group, he suggested that oxidative DNA adducts could serve as novel biomarkers of BPD and ARDS. Also, CYP1B1 plays a key role in oxygen-mediated lung injury. Therefore, the study provides novel opportunities to develop rational strategies for the prevention/treatment of hyperoxia-induced lung diseases in humans.

My overall takeaway from this Symposium stems from the presentation by Dr. Kadiiska that it is equally important to determine what did and did not work (as negative data are an important result) because each assay has a strong advocate of being the best and applicable for the measurement of oxidative stress.

This blog was prepared by an SOT Reporter. SOT Reporters are SOT members who volunteer to write about sessions and events they attend during the SOT Annual Meeting and ToxExpo. If you are interested in participating in the SOT Reporter program in the future, please email Giuliana Macaluso.

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