lec 7: Oxidative stress signaling in health and diseases

What are Reactive oxygen species (ROS)

Highly reactive molecules derived from oxygen and are generated as natural byproducts of normal cellular respiration.

most common ROS

1. singlet oxygen

2. superoxide anion

3. hydrogen peroxide

4. hydroxyl radical

Reactive nitrogen species

various nitric oxide derived compounds

sources of ROS

endogenous sources

exogenous sources

Endogenous sources of ROS

1. Mitochondrial respiration (major source): byproduct of natural respiration

2. immune cells: neutrophils and macrophages produce ROS as immune response

3. enzymatic activity: xanthine oxidase and NADPH oxidase

4. Endoplasmic reticulum

5. mental stress and ageing

Mitochondria are the powerhouses of the cell, and they produce ROS as a byproduct of ……….

ATP generation

Exogenous sources

1. pollutants

2. heavy metals

3. xenobiotics and drugs

4. cigarettes smoke

5. radiation

6. alcohol

7. food and nutrients

Importance of ROS

• cell signalling: especially in the ones that regulate cell growth/ development, diffrentiation and survival

• wound healing: ROS play a role in the wound healing process by promoting cell migration and proliferation

• cell death: reg apoptosis and autophagy

• Immune response: by immune cells to destroy pathogens and promote inflammation

ROS can activate various pathways as……. and they play important roles in …

RAS/RAF/MAPK, JAK/STAT, PI3K/AKT, NF-kB

cell signaling, development, immunity, cell survival, proliferation, and apoptosis.

increased levels of ROS =

activation of defense mechanisms

Natural defense mechanisms against ROS include:

• Antioxident enzymes: Superoxide dismutase SOD, catalase CAT, glutathione peroxidase GPX

• Antioxidant metabolites: Vit C (ascorbic acid), Vit E, Carotenoids, Flavanoids

…………. is the master regulator of gene expression of antioxident enzymes

Nuclear transcription factor Nrf2

oxidative stress

The imbalance between the production of ROS/RNA and the ability of the antioxidant defense system to neutralize them

Cellular targets of ROS that account for toxicity:

1. Lipid peroxidation: membrane integrity and fluidity

2. DNA damage: SSBs, DSBs. Unrepaired or misrepaired could lead to mutations and genomic instability

3. Protein oxidation: structural changes and loss of function, disease development

Effects of ROS leads to

cell death

DNA damage

Aging

Inflammation

DNA DAMAGE

formation of 8-oxo guanine (specific to ROS only) induces mutation in tumor suppressor genes and genome instability. Cancer and 8-oxo-G are considered markers of oxidative stress in diseases like: cardiovascular disease, neurodegenrative disease, and inflammatory diseases.

Marker of oxidative stress

8-oxoG

which protein of the PI3K kinase family detects DSBs

ATM (ataxiatelangiectasia mutated)

ATM is the main sensor of

DNA damage

what happens once ATM detects DNA damage

DNA damage response DDR is activated

Why is the DNA damage response important?

Avoid the transmission of mistaken genetic information to daughter cells and protect cells from deregulated metabolism.

steps of DDR

1. ATM ctivation activates Chk1,2

2. H2AX phosphorylation at DSB site retruit BRACA1 and 53BP1

3. at the same time, p53 accumlates and activates p21

4. activate intrinsic apoptosis pathway (pro-apoptotic BAX/BAK)

5. DNA repair through NHEJ and HRR

Function of Chk1 and 2

involved in cell cycle regulation, and arrest in case of damage

…………… and ….. are involved in the DNA repair

BRACA1 and 53BP1

p21

activated by p53, and involved in cell cycle arrest

NHEJ

NOn-homologous end joining

HRR

Homologous recombinational repair

Molecular Techniques to investigate DNA damage

8-oxoG: ELISA or Immunohisochemistry

commet assay

yH2AX assay: cells can be stained with a fluorescent antibody against γH2AX to identify cells with DNA damage.

Commet assay

Cells are embedded in agarose and then lysed to release the DNA. The DNA is then subjected to an electric field, which causes the damaged DNA to migrate further than the intact DNA. The amount of DNA migration is then measured to quantify the amount of DNA damage in the cells.

CELL DEATH

ROS may induce apoptosis, necrosis or autophagy

• p53 pathway: in response to DNA damage (intrinsic)

• TNF-TNFR: NF-KB pathway activation, upreg TNF-TNFR inflammatory pathway (extrinsic)

• Autophagy and necrosis: due to mitochondrial dysfunction, peroxidation, and inflammation.

Molecular Techniques to investigate cell death

Annexin V- PI assay

Tunnel assay

caspase gene/protein level

INFLAMMATION

NF-KB signaling pathway: major regulator of inflammatory gene expression

MAPK signaling pathways

Cyclooxygenase (COX)

Cycloooxygenase enzyme

involved in the synthesis of pro-inflammatory prostaglandlins

…………….. is the major regulator for the inflammatory gene expression, while ….. is the major regulator for antioxidant enzyme gene expression

NF-KB, Nrf2

Medications for targeting inflammatory pathways

1. NSAIDS: such as ibuprofen and celecoxib inhibit COX enzymes

2. Corticosteroids: supress NF-KB and other inflammatory pathways

3. infliximab: chimeric monoclonal antibody (remicade), TNF-alpha inhibitor for autoimmune diseases

Molecular Techniques to investigate Inflammation

Measuring the level of the inflammatory markers (e.g., IL-6, IL-1β, TNF-α, TGF-β) by:

❑ Reverse Transcription Quantitative PCR (RT-qPCR)

❑ Gene Expression arrays (multiple markers)

❑ Enzyme-Linked Immunosorbent Assay (ELISA)

❑ Western blot

❑ Immunohistochemistry

❑ Protein Arrays (multiple markers)

CELL AGING

also known as cellular senescence.

it can be accelerated in reponse to stressors such as ROS, inflammation and DNA damage

Pathways related to cell aging

DNA damage: lead to p53 accumlation, cellular senescence

MAPK/ERK: indirectly activate senescence

Telomerase shortening: accelerate telomerase shortening

What happens when telomeres get critically short

cells may enter a state of replicative senescence, leading to aging and functional decline

cellular senescence

is a state in which cells undergo permanent cell cycle arrest and undergo distinctive phenotypic and functional changes (stays metabolically active).

SASP

secreted by senescent cells, called senescence-associated secretory phenotype, which are bioactive molecules such as pro-inflammatory cytokines, proteases, growth factors, and chemokines.

Molecular Techniques to investigate Cellular Senescence

• senescence associated beta galactosidase

• detect SASP by ELISA, western blot or multiplex assays

• Telomere length: qPCR and q-FISH

Senescence-Associated β-Galactosidase (SA-β-Gal)

widely used biomarker for detecting senescent cells. It is based on the activity of β-galactosidase at pH 6.0, which is upregulated in senescent cells, resulting in a blue-green product that can be detected by microscopy or fluorescent filter in Plate reader

Molecular Techniques to investigate ROS

Molecular Techniques to investigate ROS

• Fluroscent 2',7'-Dichlorofluorescein Diacetate (DCF-DA) Assay: fluorescent microscop/plate read

• Hydrogen peroxide fluroscent sensors

• nuclear factor erythroid 1- related factor 2: Nrf2 activation assays : western blot/ qPCR

• Mitochondrial ROS detection (MitoSOX)