Diminished protective oxidative stress response

The "Diminished Protective Oxidative Stress Response" is a critical key event in the Adverse Outcome Pathway (AOP) framework that plays a central role in understanding how exposure to various stressors can lead to adverse outcomes.

Oxidative stress is caused by an imbalance between the production of reactive oxygen and the detoxification of reactive intermediates. Reactive intermediates such as peroxides and free radicals can be very damaging to many parts of cells such as proteins, lipids, and DNA. Severe oxidative stress can trigger apoptosis and necrosis. (Ref. IPA, NRF2-mediated Oxidative Stress Response, version60467501, release date: 2020-11-19)

One essential component of this key event is the activity of Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that plays a pivotal role in the regulation of the oxidative stress response. Detecting Nrf2 activity is crucial for assessing the status of the oxidative stress response.

The cellular defence/defense response to oxidative stress includes induction of detoxifying enzymes and antioxidant enzymes. Nuclear factor-erythroid 2-related factor 2 (Nrf2) binds to the antioxidant response elements (ARE) within the promoter of these enzymes and activates their transcription. Inactive Nrf2 is retained in the cytoplasm by association with an actin-binding protein Keap1. Upon exposure of cells to oxidative stress, Nrf2 is phosphorylated in response to the protein kinase C, phosphatidylinositol 3-kinase and MAP kinase pathways. After phosphorylation, Nrf2 translocates to the nucleus, binds AREs, and transactivates detoxifying enzymes and antioxidant enzymes, such as glutathione S-transferase, cytochrome P450, NAD(P)H quinone oxidoreductase, heme oxygenase, and superoxide dismutase. (Ref. IPA, NRF2-mediated Oxidative Stress Response, version60467501, release date: 2020-11-19)

Nrf2, a master regulator of oxidative stress through enhanced expression of anti-oxidant genes of glutathione and thioredoxin-antioxidant systems, has anti-inflammatory, anti-apoptotic, and antioxidant effects. Dimethyl fumarate (DMF), an activator of Nrf2, can decrease inflammation and reactive oxygen species (ROS) through the inhibition of NF-kappaB by inducing anti-oxidant enzymes (Jackson et al, 2014; Hassan et al, 2020; Timpani et al, 2021).

Inactivation of Nrf2 causes diminished protective responses to ROS.

Oxidative stress can be measured as follows:

1. Direct detection of reactive oxygen species (ROS)

ROS can be detected by intracellular ROS assay, in vitro ROS/RNS assay. Nitric oxide can be detected in intracellular nitric oxide assay (Ashoka et al, 2020).

Hydroxyl, peroxyl, or other ROS can be measured using a fluorescence probe, 2', 7'-Dichlorodihydrofluorescin diacetate (DCFH-DA), at fluorescence detection at 480 nm/530 nm.

Hydrogen peroxide (H2O2) can be detected with a colorimetric probe, which reacts with H2O2 in a 1:1 stoichiometry to produce a bright pink colored product, followed by the detection with a standard colorimetric microplate reader with a filter in the 540-570 nm range.

ROS can be detected by PEGylated bilirubin-coated iron oxide nanoparticles in whole blood (Lee et al, 2020).

2. Measurement of anti-oxidants

The level of catalase, glutathione, or superoxide dismutase can be measured as anti-oxidants. Catalase is an anti-oxidative enzyme that catalyses the resolution of hydrogen peroxide (H2O2) into H2O and O2. The chemiluminescence or fluorescence of HRP catalytic reaction can be detected with residual H2O2 and probes (DHBS+AAP, or ADHP (10-Acetyl-3, 7-dihydroxyphenoxazine)).

Anti-oxidant capacity is also one of the oxidative stress markers. Oxygen radical antioxidant capacity (ORAC), hydroxyl radical antioxidant capacity (HORAC), total antioxidant capacity (TAC), the cell-based exogenous antioxidant assay can be used for measuring the antioxidant capacity.

3. Detection of damages in protein, lipid, DNA or RNA

Oxidation of protein can be measured by the detection of protein carbonyl content (PCC), 3-nitrotyrosine, advanced oxidation protein products, or BPDE protein adduct. 

DNA oxidation can be detected with 8-oxo-dG / 8-hydroxy-2'-deoxyguanosine (8-OHdG) by ELISA or HPLC (Chepelev et al, 2015; Valavanidis et al, 2009).

Lipid peroxides decompose to form malondialdehyde (MDA) and 4, hydroxynonenal (4-HNE), natural bi-products of lipid peroxidation. Lipid peroxidation can be monitored by thiobarbituric acid (TBA) reactive substances in biological samples. MDA and TBA form MDA-TBA adduct in a 1:2 stoichiometry and detected by colorimetric or fluorometric measurement.

4. Detection of Nrf2 activity

Measuring Nrf2 activity involves assessing its transcriptional activity or protein abundance.

Several methods can be employed to detect Nrf2 activity, and these include:

a. Luciferase Reporter Assay:

   - This widely used method involves creating a reporter plasmid containing Nrf2-responsive antioxidant response element (ARE) sequences and a luciferase gene.

   - Cells of interest are transfected with the Nrf2-ARE luciferase reporter construct.

   - After exposure to the stressor of interest, cells are lysed, and luciferase activity is measured.

   - Increased luciferase activity indicates Nrf2 activation, while decreased activity suggests diminished Nrf2 activity.

b. Quantitative PCR (qPCR):

   - Assessing Nrf2 activity at the transcriptional level can be achieved through qPCR.

   - Specific Nrf2 target genes (e.g., NQO1, HO-1) are selected and their mRNA levels are quantified.

   - Increased expression of these genes is indicative of Nrf2 activation, while reduced expression suggests diminished Nrf2 activity.

c. Western Blotting:

   - This method allows the detection of Nrf2 protein levels in cell or tissue samples.

   - After exposure to a stressor, proteins are extracted, separated by electrophoresis, and transferred to a membrane.

   - Specific antibodies against Nrf2 are used to detect its abundance.

   - Increased Nrf2 protein levels suggest Nrf2 activation, while reduced levels indicate diminished activity.

d. Immunofluorescence:

   - Immunofluorescence can be used to assess the cellular localization of Nrf2.

   - Cells are fixed and probed with antibodies specific to Nrf2, followed by fluorescently labeled secondary antibodies.

   - Nrf2 localization within the cell (e.g., cytoplasm or nucleus) can indicate its activation status.

e. Electrophoretic Mobility Shift Assay (EMSA):

   - EMSA is a technique that measures the binding of Nrf2 to ARE sequences.

   - Radioactively labeled ARE sequences are incubated with nuclear extracts, and the formation of DNA-protein complexes is visualized on a gel.

   - The intensity of the complex can indicate Nrf2 binding activity.  

Response to ROS occurs in many cell types and tissues in all life stages and the broad range of mammals.

Ashoka, A.H. et al. (2020), “Recent Advances in Fluorescent Probes for Detection of HOCl and HNO”, ACS omega, 5(4), 1730-1742. https://doi.org/10.1021/acsomega.9b03420.

Chepelev, N.L. et al. (2015), “HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, in Cultured Cells and Animal Tissues”, J Vis Exp, e52697-e52697, https://doi.org/10.3791/52697.

Hassan, S.M. et al. (2020), “The Nrf2 Activator (DMF) and Covid-19: Is there a Possible Role?”, Med Arch, 74(2), 134-138. https://doi.org/10.5455/medarh.2020.74.134-138.

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”, Toxicol Appl Pharmacol, 274, 63-77, https://doi.org/10.1016/j.taap.2013.10.019.

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. https://doi.org/10.7150/thno.39662.

Timpani, C.A, E. Rybalka. (2021), “Calming the (Cytokine) Storm: Dimethyl Fumarate as a Therapeutic Candidate for COVID-19.”, Pharmaceuticals, 14(1), 15. https://doi.org/10.3390/ph14010015.

Valavanidis, A. et al. (2009), “8-hydroxy-2' -deoxyguanosine (8-OHdG): A critical biomarker of oxidative stress and carcinogenesis”, J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 27, 120-39. https://doi.org/10.1080/10590500902885684