Biochem Exam 2 Lecture 1

Name four major sources of ROS

1. Cellular respiration (mitochondria). 2. Spontaneous (Fenton reaction). 3. Enzymatic reactions (NADPH oxidase): Our neutrophils use this enzymatic complex to generate ROS to kill microorganisms. 4. Ionizing radiation: When our bodies are hit with radiation, these particles will damage our DNA and make ROS. 80% of damage to our DNA comes from the ROS.

List 9 diseases and disorders in which ROS have been implicated.

1. Chronic granulomatous disease which is a defect in NADPH oxidase, so people cannot use it in the phagocytes/neutrophils, so we get a suppression of the immune system. 2. Parkinson's disease, excess ROS production in the neurons of your brain that is rich in dopamine metabolism. 3. Ischemia/hypoxia reperfusion. It is for your heart/brain, so you cut off blood flow and high amounts of ROS. 4. Oxygen toxicity (pulmonary and CNS). 5. Epilepsy, disregulation of redox balance in the brain. 6. Cancer, where the mitochondria is defective. 7. Diabetes. Higher levels of ROS. 8. Alzheimer's disease. 9. ALS.

What does ROS do to DNA damaging, change in signaling, protein oxidation, and lipid peroxidation?

1. DNA damage: increases cell death (oncosis, apoptosis), decreases proliferation. 2. Signaling: trascription factor activation, decreases NO signaling → decreases angiogenesis (physiological process through which new blood vessels form from pre-existing vessels), neuroexcitotoxicity. 3. Protein oxidation: decrease in enzymatic functions, growth factor inhibition. 4. Lipid peroxidation: prostanoids → inflammation, lipid chain reaction → radical formation.

How do ROS and RNS cause Parkinson's disease?

1. Dopamine levels are reduced by MAO, which generates H₂O₂. 2. Superoxide also can be produced by mitochondria, which SOD will convert to H₂O₂. Iron levels increase, which drives Fenton reaction. 3. NO (from NOS) reacts with superoxide to form RNS. 4. The RNS and hydroxyl radical lead to radical chain reactions that result in lipid peroxidation, protein oxidation, the formation of lipofuscin, and neuronal degeneration. A ROS-induced mechanism of neurodegeneration in Parkinson's disease has been shown using MPTP

What are three dietary antioxidants?

1. Resveratrol appears to mimic several of the biochemical effects of calorie restriction. Some studies indicates that resveratrol activates Sirtuin 1 (SIRT1) and PGC-1α and improve functioning of the mitochondria. 2. EGCG - along with other flavonoids - can be beneficial in treating brain, prostate, cervical and bladder cancers. 3. Beta carotene in the diet of flamingoes (blue-green algae) give them a pink color.

What are four examples of exogenous antioxidants?

1. Vitamin C (ascorbate) 2. Vitamin E (tocopherols, tocotrienols) 3. Polyphenolic antioxidants (resveratrol and flavonoids). Coffee has a lot of flavonoids. 4. Carotenoids (lycopene, carotenes, lutein)

What is a ROS?

A reactive oxygen-containing radical (unpaired electron) or a reactive molecule that generates a radical. It is a consequence of normal cellular metabolism.

What are the two enzymatic defenses against ROS?

A) Superoxide dismutase (SOD) and catalase (CAT) are both highly specific for their substrates, O₂• and H₂O₂, respectively. B) Glutathione peroxidase (GPx) reduces H₂O₂ and lipid peroxides (LOOH), using GSH as a co-substrate. THe GSH is recycled by gutathione reductase (GR) using NADPH from the pentose phosphate pathway. C) Structure of GSH, which is comprised of glycine, cysteine, and glutamate.

Explain the relationship with ALS and superoxide dismutase (SOD)

ALS normally comes from an overproduction of SOD. A mutation in SOD is responsible for familial cases of ALS (about 10%). Three forms of SOD are present in humans: 1. SOD1 (CuZnSOD) in the cytoplasm. 2. SOD2 MnSOD in the mitochondria. 3. SOD3 (CuZnSOD) is extracellular.

What is respiratory burst?

Also known as oxidative burst. Neutrophils (or phaogcytes) give in response to attacking microorganisms, is the rapid release of ROS by phagocytosis.

What does catalase do specifically for H₂O₂

Catalyzes the decomposition of hydrogen peroxide to water and oxygen. Located in peroxisomes. Highest turnover numbers of all enzymes.

What is the relationship of enhanced ROS production and tumor progression?

Enhanced ROS production drives tumor progression. Increased basal ROS production makes cancer cells vulnerable to chemotherapeutic agents that further augment ROS production. Antioxidant inhibitor (PEITC) elevates ROS by inhibiting the glutathione peroxidase and GSH. Tumor cells are killed by the same redox system they require for growth.

Summary 4

Enzyme-based cellular defense mechanisms include SOD, catalase and glutathione peroxidase. Vitamins C and E play important roles by arresting chain reactions started by ROS. Explain how chemical or environmental triggers (e.g. ionizing radiation) can result in excess ROS production, mitochondrial damage and cancer.

What is Glutathione reductase? How does it work with Glutathione peroxidase?

Glutathione reductase regenerates reduced glutathione. GSH peroxidase has selenocysteine in its active site. An increased GSSG to GSH ratio is considered an indicative of oxidative stress.

There are two physiological functions of ROS redox signaling. What are they?

Hypoxic pulmonary vasoconstriction and hypoxic ventilatory response

Explain the mechanism of hypoxic pulmonary vasoconstriction

In normoxia, the proximal mitochondrial ETC produces ROS, some of which (such as H₂O₂) can diffuse to the plasma membrane, oxidizing and thus opening K⁺ channels (this happens under normal conditions). This favors normoxic membrane hyperpolarization and vasodilatation. Hypoxia is sensed in the proximal ETC, and perhaps vascular oxidases, resulting in a decrease in ROS production. Decrease in ROS levels (reduced state) that causes K⁺ channel inhibition (closing), membrane depolarization (from decreased K⁺ efflux), and vasoconstriction. This process is a physiological response to hypoxia (e.g. high altitude). There is an increase in ventilation and redistribution of blood flow to maintain tissue oxygenation.

Biological importance of superoxide anion (O2•)

Intracellular ROS produced at the ETC and other sites. Precursor to more reactive species. Spontaneous formation to H₂O₂.

How are ROS generated from radiation?

Ionizing radiation consists of subatomic particles or electromagnetic waves energetic enough to detach electrons from H2O or O2, thus ionizing them. Radiation-induced ROS production is proportional to O2 concentration.

What is hypoxic ventilatory response?

It is a physiological function of ROS redox signaling. Hypoxic inhibition of K⁺ current causes rapid and reversible depolarization of carotid body glomus cells, pulmonary vasculature and specific populations of central neurons. VR is the increase in ventilation induced by hypoxia. It is initially elevated in lowland people who go to high altitude, although it reduces significantly over time as people acclimatize

What is hypoxic pulmonary vasoconstriction?

It is a physiological function of ROS redox signaling. Paradoxical, physiological phenomenon in which pulmonary arteries constrict in the presence of hypoxia (low oxygen levels), redirecting blood flow to alveoli with a higher oxygen content. Lungs pick up more oxygen via hemoglobin.

How does the mitochondria produce ROS?

It is as a side-reaction during oxidative phosphorylation. After the oxidation of NADH, the electron transport chain catalyzes single-electron redox reactions. Mitochondria, which harbor the bulk of oxidative pathways, are packed with various redox carriers that can potentially leak single electrons to oxygen and convert it into superoxide anion, a progenitor ROS.

What is the Haber-Weiss Reaction?

It is catalyzed by metals. There are physilogical implications because this is always happening in our tissue. It generates •OH (hydroxyl radicals) from H2O2 (hydrogen peroxide) and superoxide (•O2-). The reaction is very slow, but it is catalyzed by iron. The first step of the catalytic cycle involves the reduction of ferric (Fe³⁺) iron to ferrous (Fe²⁺) iron

What is a reactive nitrogen species?

It is generated enzymatically from nitric oxide synthase. It is also generated from pollutants like smog and cigarette smoke.

What is ROS production proportional to?

It is proportional to tissue pO₂ and excess ROS is associated with oxygen toxicity.

What does Glutathione peroxidase do? What is Glutathione?

It reduces H₂O₂ to water. Glutathione is comprised of 3 amino acids; glycine, cysteine, glutamate. The sulfhydryl group of glutathione, which is oxidized to a disulfide. Glutathione peroxidase transfers electrons from glutathione (GSH) to hydrogen peroxide. It plays a role in detoxification of your liver

Explain the antioxidant activity of ascorbate

L-ascorbate exists as an anion at physiological pH. Spontaneously, it reduces radicals, forming a dehydroascorbyl radical (As∙). Dehydroascorbate, formed by a second reduction reaction or by a dismutation reaction, is recycled by dehydroascorbate reductase (GSH-dependent enzyme). Dehydroascorbyl radical may also dismutate to ascorbate and dehydroascorbate.

How can NADPH oxidase and ROS be beneficial to a cell?

NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) is a membrane-bound enzyme complex. It can be found in the plasma membrane as well as in the membrane of phagosome. It is made up of six subunits (Rho GTPase and 5 Phox). The complex is latent in neutrophils and is activated to assemble in the membranes during respiratory burst. Superoxide dismutase helps form hydrogen peroxide. Myeloperoxidase and Cl- can help form HOCL- from hydrogen peroxide. ROS production during phagocytosis is initiated through NADPH oxidase and kills invading organisms. Hydrolytic enzymes are also released from lysosomes to assist in degradation of microbial debris.

How is the NADPH oxidase complex get activated? Why is this important for ROS?

NADPH oxidase complex is latent until activated (PKC-dependent) to assemble the 6 subunits in the membrane. This is activated by calcium. NADPH oxidase generates ROS by transferring electrons from NADPH inside the cell across the membrane and coupling these to O2 to produce superoxide (O2•).

How can nitric oxide be regulated?

NO can be regulated by regulating arginine and citruline or through enzymes. Arginine converts to citruline. Nitric oxide is a powerful vasodialator (widening of blood vessels).

Biological importance of hydrogen peroxide (H₂O₂)

Not a free radical, but can generate free radicals by reaction with transition metals (e.g. Fe²⁺). Can diffuse into and through cell membranes.

Summary 2

Oxidative stress occurs when ROS species are produced faster than can be accommodated by cellular defense mechanisms. ROS are associated with a variety of pathologies.

What molecule is the precursor for our first ROS? What happens after?

Oxygen is the precursor to our first ROS. Next, we have the biradical, which is the natural, ground-state form of O₂ at body temperature. Then, we will have the superoxide anion as the first ROS generated (O₂•). Reduction of the superoxide yields to hydrogen peroxide, H₂O₂. Reduction of hydrogen peroxide releases a hydroxyl radical (OH•) and a hydroxide ion (OH⁻). Metals like copper are needed to make the hydroxyl radical. Water is the end product of complete reduction of O₂.

Biological importance of nitric oxide (NO)

Produced by NOS. Combines to O₂ or other oxygen-containing radicals to produce additional RNS.

Biological importance of hypochlorus acid (HOCl)

Produced in neutrophils during respiratory burst to destroy invading organisms

Summary 3

ROS damage membrane lipids. PUFAs are very susceptible to peroxidation by free radical-based chain reactions. Proteins, nucleic acids and carbohydrates are also modified by ROS

What is chronic granulomatous disease?

Rare, inherited disorder (X-linked; 1 in 200,000 in US). Characterized by defective phagocytes (white blood cells, neutrophils). Patients vulnerable to severe recurrent bacterial and fungal infections. Defect in component of NADPH oxidase (respiratory burst oxidase). Failure to produce superoxide radicals (O₂•). Granulomas form at sites of infection.

Summary 1

Reactive oxygen species (ROS) include superoxide (O2.-), hydrogen peroxide (H2O2), and hydroxyl radical (•OH). ROS can be generated as by-products of the ETC, spontaneously, enzymatically and by ionizing radiation.

Biological importance of peroxyl radical (LOO•)

Released during membrane lipid peroxidation

What happens if we have ROS damage?

Reperfusion injury aka reoxygenation. It is the most severe in tissues with high O₂ demands like the heart and brain.

What is retrograde response?

Retrograde Response (RTG): Mitochondrial retrograde signaling is a pathway of communication from mitochondria to the nucleus that influences many cellular and organismal activities under both normal and pathophysiological conditions.

How does lipid peroxidation occur?

Starts with hydroxyl radical that reacts with an unsaturated lipid (omega 6 or omega 3). Forms a lipid radical. In the presence of oxygen, we can have a lipid peroxyl radical. You need an antioxidant to terminate this (vitamin E). When a radical reacts with a non-radical, it always produces another radical, which is why the process is called a "chain reaction mechanism".

Biological importance of peroxynitrite (ONOO⁻)

Strong oxidizing and nitrating agent.

Explain ROS-mediated cell injury

Superoxide and the hydroxyl radical initiate lipid peroxidation in the cellular, mitochondrial, nuclear, endoplasmic reticulum membranes. The increase in cellular permeability results in an influx of Ca²⁺, which causes further mitochondrial damage. The cysteine sulfhydryl groups and other amino acid residues on proteins are oxidized and degraded. Nuclear and mitochondrial DNA can be oxidized, resulting in strand breaks and other types of damage. RNS (NO, NO2, and peroxynitrite) have similar effects.

What is the Fenton Reaction?

The Fenton reaction is a reaction between ferrous (Fe²⁺) iron and hydrogen peroxide. We produce a ferric (Fe³⁺) iron and the hydroxyl radical. This is the most damaging ROS in the biological system. Can be seen in membrane lipid peroxidation. Cu⁺ catalyzes the same reaction.

Explain the metabolic theory of cancer

The metabolic theory of cancer is based on the observation that cancers are primarily a disease of mitochondrial origin with 90-95% of cases attributed to environmental factors (targeting the mitochondria) and only 5-10% due to genetics. Progressive mitochondrial dysfunction from chemical or environmental triggers leading to genomic instability and activation of oncogenes. Defective Mitochondria cause excess ROS production which drives tumor progression

Biological importance of hydroxyl radical (•OH)

The most reactive species in attacking biological molecules. Produced from H₂O₂ in the Fenton reaction in the presence of Fe²⁺ or Cu⁺.

During oxidative phosphorylation, how does Q make radicals?

The semi-quinone radical (Q) is an intermediate in the reduction of Q to QH₂ by complex I or II. It is sensitive to oxidation by molecular oxygen and is considered a major source of superoxide radicals in the cell. This reaction is enhanced by hyperoxia (excess oxygen in the cell)

What is reoxygenation?

The sudden reintroduction of O₂ that generates ROS

What happens during ischemia/hypoxia reperfusion injury?

The sudden reintroduction of molecular oxygen causes re-energization of mitochondria and reactivation of the electron transport chain with massive production of ROS, which may stimulate further ROS production (ROS-induced ROS release) and generation of RNS in the presence of NO. ATP production restored, the activity of the Na⁺/Ca²⁺ exchanger is restored, leading to the extrusion of Na⁺ in exchange for Ca²⁺ → cytosolic Ca²⁺ overload. Ca²⁺ accumulation in the mitochondrial matrix, ROS/RNS favors the formation/opening of the mPTP favoring necrotic cell death, most likely in those cells that have already sustained injury during ischemia.

What is so important about Trolox?

Trolox is an analog to vitamin E. It is important for stopping the chain reaction of membrane-lipid oxidation.

When you have a tumor, what happens to their blood supply?

Tumors, when they grow, outgrow their blood supply so they become hypoxic

Describe the antioxidant activity of vitamin E

Vitamin E (tocopherol and tocotrienol isomers) is a lipophilic antioxidant that prevents membrane lipid peroxidation. α-Tocopherol is the most effective form of vitamin E in the diet. Tocopherols reduce lipid hydroperoxyl radicals and also inactivate singlet oxygen. The tocopheryl radical is recycled by ascorbate. Tocopheryl quinone (oxidative metabolite) is also formed in small quantities. Taking vitamin E and vitamin C are synergistic so the two should be taken together.

What is the Warburg affect?

Warburg effect is the observation that cancer cells rely primarily on glucose (glycolysis) and substrate levels phosphorylation (SLP) for energy due to defective mitochondria. This explains why calorie restriction diets and ketogenic diets (very low carbohydrate diets) prevent the progression of cancer.

What are the effects of ROS on our mitochondria?

Your mitochondria is a source of the 1-2% oxygen we use for reactive oxygen species (superoxide anion). As we age, our mitochondria makes more reactive oxygen species. We tend to produce more oxidative stress. High levels of oxidative stress are damaging to the part of our cells that make energy like ATP. Free radicals tend to put a hit on mitochondrial function.