Diminished protective oxidative stress response

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)

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.

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