04. Free Radicals

What are 5 antioxidant defenses?

- SOD - Catalase - Glutathione peroxidase - Vitamin C - Vitamin E

Catalase removes ____ decomposing it to water and O2. Catalase is present in all major body organs being especially concentrated in ____ Catalase activity is concentrated on ____ Catalase consists of 4 protein subunits: each subunit containing a ____ heme group bound to its active site.

Catalase removes hydrogen peroxide (H2O2) decomposing it to water and O2. Catalase is present in all major body organs being especially concentrated in liver. Catalase activity is concentrated on peroxisomes. Catalase consists of 4 protein subunits: each subunit containing a ferric Fe3+ heme group bound to its active site. Note: catalase will also remove H2O2 when it is formed from SOD

What is a free radical? Free radicals are produced when the _____ bond is broken and one electron from each pair remains with each atom. A free radical is an unstable configuration and therefore highly reactive, owing to the tendency of electrons to pair (i.e. tendency of free radicals to donate or accept one electron). A free radical lasts just few (seconds, minutes, hours, or days), because it reacts with adjacent molecules such as proteins, nucleic acids or lipids. When a free radical reacts with a _____ molecule, a _____ reaction is initiated, whereby these molecules are themselves converted into free radicals The chain reaction is terminated when ____ react with each other and cross-link two unpaired electrons forming a covalent bond

Chemical species with an unpaired electron in the outer orbit Free radicals are produced when the covalent bond is broken and one electron from each pair remains with each atom. A free radical is an unstable configuration and therefore highly reactive, owing to the tendency of electrons to pair (i.e. tendency of free radicals to donate or accept one electron). A free radical lasts just few milliseconds, because it immediately reacts with adjacent molecules such as proteins, nucleic acids or lipids. When a free radical reacts with a non-radical molecule, a chain reaction is initiated, whereby these molecules are themselves converted into free radicals The chain reaction is terminated when two free radicals react with each other and cross-link two unpaired electrons forming a covalent bond, each radical contributing its single unpaired electron.

Carbonylation and sulfonation cause protein ____ and loss of normal function. Carbonylation and sulfonation make proteins more sensitive to _____, compared to the non-oxidized.

Carbonylation and sulfonation cause protein misfolding and loss of normal function. Carbonylation and sulfonation make proteins more sensitive to proteolysis, compared to the non-oxidized - When the rate of protein proteolysis is higher than the rate of protein synthesis, permanent cellular damage and cell death are observed.

Sources of free radicals: ______ enzymes of the ER are involved in the oxidation of a wide range of substrates at the expense of oxygen Oxidation is the combination of oxygen with a substance forming oxide. _____ are capable of reducing oxygen to superoxide before using it in oxidizing operations. How does this generate free radicals?

Cytochrome P450 enzymes of the ER are involved in the oxidation of a wide range of substrates at the expense of oxygen Oxidation is the combination of oxygen with a substance forming oxide. Oxidases are capable of reducing oxygen to superoxide before using it in oxidizing operations. Some superoxide always diffuses away from the enzyme before it is used in the chemical reaction.

Sources of free radicals: Describe how free radicals are formed in the mitochondria. What occurs because of free radical damage in the mitochondria?

During the energy transduction in the electron chain, a small number of electrons (about 3%) leak to oxygen prematurely forming the radical superoxide --> Superoxide is released in mitochondrial matrix. Mitochondrial lipids damaged by free radical reactions form cross-linked compounds that accumulate in the form of yellow-brown pigment granules (lipofuscin). - note the yellow/gold = lipofuscin

During the respiratory burst from PMN's --> the extra O2 is not related to mitochondrial respiration, but is accomplished by the enzyme _____, a protein complex that assembles in the wall of the phagocytic vacuole and produces _____ in the vacuole. Activation of NADPH during phagocytosis of bacteria involves migration of cytosolic components to the _____ to assemble the active complex.

During the respiratory burst from PMN's --> the extra O2 is not related to mitochondrial respiration, but is accomplished by the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a protein complex that assembles in the wall of the phagocytic vacuole and produces superoxide in the vacuole. Activation of NADPH during phagocytosis of bacteria involves migration of cytosolic components to the phagosome membrane to assemble the active complex.

Free radicals directly attack proteins and oxidize them. There are two unfavorable effects of protein peroxidation: •Modification of amino acids CH2 side chains to ____ known as ______ •Oxidation of sulfur groups of sulfur-containing amino acids methionine and cysteine to specific forms --> known as ______

Free radicals directly attack proteins and oxidize them. There are two unfavorable effects of protein peroxidation: •Modification of amino acids CH2 side chains to carbonyl (C=O) known as carbonylation •Oxidation of sulfur groups of sulfur-containing amino acids methionine and cysteine to specific forms --> known as sulfonation

Glutathione peroxidase (GPx). GPx is a family of enzymes that remove ____ and ____. So far 8 isoforms of GPx have been identified. GPx1 is the most abundant form in the ____. It removes H2O2 produced in ____and ____. GPx4 removes ____. GPx contains metal ions (____) in its active site.

Glutathione peroxidase (GPx). GPx is a family of enzymes that remove hydrogen peroxide and lipid hydroperoxides. So far 8 isoforms of GPx have been identified. GPx1 is the most abundant form in the cytoplasm. It removes H2O2 produced in mitochondria and ER. GPx4 removes lipid hydroperoxides. GPx contains metal ions (selenium) in its active site.

Sources of free radicals: What is the Fenton reaction?

H2O2 + Fe2+ --> OH` + Fe3+

H2O2 is a (weak or strong) oxygen radical H2O2 can directly damage ____ (inactivate enzymes) but has no direct effect on _____.

H2O2 is a weak oxygen radical H2O2 can directly damage proteins (inactivate enzymes) but has no direct effect on DNA.

What ROS is the most dangerous?

Hydroxyl radical (OH`)

What three things can Hydroxyl radical (OH˙) damage>

Hydroxyl radical can directly damage proteins (inactivate enzymes), trigger lipid peroxidation and damage DNA

Hypochlorous acid (HOCl) is produced by the enzyme _____ of the azurophilic granules of neutrophils and is bactericidal --> Neutrophils use HOCl to kill bacteria

Hypochlorous acid (HOCl) is produced by the enzyme myeloperoxidase (MPO) of the azurophilic granules of neutrophils and is bactericidal --> Neutrophils use HOCl to kill bacteria

What are the three biomarkers for lipid peroxidation? What is the best one?

IPs, MDA, and HNE IPs are the best one because their levels are not affected by diet.

Review: which free radicals can attack DNA? The _____ base is highly susceptible to oxidative stress

Only hydroxyl radicals and peroxynitrite can attack DNA The guanine base is highly susceptible to oxidative stress

The best biomarker for oxidative DNA damage is _____ --> it evaluates the ____ of ROS-forming chemicals

The best biomarker for oxidative DNA damage is 8-oxoG --> it evaluates the cardiogenicity of ROS-forming chemicals

The biggest source of free radicals is in ______

The biggest source of free radicals is in our bodies

The ____ assay is a general assay for oxidative protein damage

The carbonyl assay is a general assay for oxidative protein damage

A free radical attack on C8 of guanine generates _____ --> adopts either an anti or syn conformation. When 8-oxoG assumes its anti conformation, it pairs with _____ , but when 8-oxoG assumes the syn conformation, it is particularly mutagenic because mimic _____ and has a strong tendency to form a base pair with _____

A free radical attack on C8 of guanine generates 8-oxoG. The 8-oxoG adopts either an anti or syn conformation. When 8-oxoG assumes its anti conformation, it pairs with cytosine, but when 8-oxoG assumes the syn conformation, it is particularly mutagenic because mimic thymine and has a strong tendency to form a base pair with adenine. - During DNA replication, DNA polymerases incorporate adenine opposite 8-oxoG. The 8-oxoG-Adenine base pairs cause GC->TA transversion mutations

After phagocytizing a bacterium, the neutrophil cell undergoes a _____

After phagocytizing a bacterium, the neutrophil cell undergoes a respiratory burst

In cell membranes, the target of free radicals is the carbon between two _____. The free radical abstracts the hydrogen atom and a lipid radical ____ is formed. Oxidation of lipid radical generates a _____ radical

In cell membranes the target of free radicals is the carbon between two double bonds of polyunsaturated fatty acid. The free radical abstracts the hydrogen atom and a lipid radical ('CH) is formed. Oxidation of lipid radical generates a lipid peroxyl radical (CH-OO˙) - Lipid peroxyl radical is the chain-carrying species that propagates the cycle.

Sources of free radicals: What are two common transition metals that can commonly produce free radicals? Why?

Iron (Fe) and Copper (Cu) --> Transition metals such as Fe and Cu can undergo autooxidation and change valence --> potentially dangerous since they catalyze unwanted free radical formation

Sources of free radicals: Iron exists into two states: ____ state (Fe2+) and ____ state (Fe3+). Copper also exists into two states: _____ (Cu2+) and _____ (3+) state.

Iron exists into two states: ferrous state (Fe2+) and ferric state (Fe3+). Copper also exists into two states: cuprous (Cu2+) and cupric (3+) state.

Isoprostanes (IPs)are produced from the free radical-catalyzed peroxidation of _____ in tissues without the direct action of cyclooxygenase (COX) enzymes. What is more toxic, MDA or IPs? What does it attack (2) ? What is the overall affect of lipid peroxidation?

Isoprostanes (IPs) are produced from the free radical-catalyzed peroxidation of arachidonic acid in tissues without the direct action of cyclooxygenase (COX) enzymes. MDA is more toxic and can attack DNA and proteins Cell membranes lose their integrity --> cells lose viability

NADPH oxidase generates superoxide by transferring _____ from cytosolic components of NADPH across the membrane of the phagocytic vacuole and coupling these to molecular oxygen to produce high amounts of superoxide anion within the phagosome. Hydrogen ions accumulate in the phagosome via channels and convert superoxide to _____. These events, which occur within minutes, initiate bacterial killing. In the presence of _____, hydrogen peroxide converts into hydroxyl radical The azurophilic granules in neutrophil cytoplasm empty their content into the phagosome. The enzyme myeloperoxidase (MPO) stored in azurophilic granules oxidizes chloride ion (Cl¯) into _____

NADPH oxidase generates superoxide by transferring electrons from cytosolic components of NADPH across the membrane of the phagocytic vacuole and coupling these to molecular oxygen to produce high amounts of superoxide anion within the phagosome. NADPH + 2O2 ↔ NADP+ + 2O2− + H+ Hydrogen ions accumulate in the phagosome via channels and convert superoxide to hydrogen peroxide. These events, which occur within minutes, initiate bacterial killing. In the presence of bacterial iron, hydrogen peroxide converts into hydroxyl radical (Fenton reaction). The azurophilic granules in neutrophil cytoplasm empty their content into the phagosome. The enzyme myeloperoxidase (MPO) stored in azurophilic granules oxidizes chloride ion (Cl¯) into hypochlorous acid (HOCl)

Nitric oxide (NO) is a signaling molecule generated by the ____ cells, _____ and _____. NO is also a powerful ____ NO reacts with superoxide to produce the damaging oxidant _____.

Nitric oxide (NO) is a signaling molecule generated by the endothelial cells, neurons and macrophages. NO is also a powerful vasodilator NO reacts with superoxide to produce the damaging oxidant peroxynitrite (ONOO˙)

Nitric oxide (NO) is generated in cells by the enzyme _____ which metabolize _____ to _____ with the formation of NO NO relaxes _____ cells in arterioles during systole, with resultant _____ . NO serves as an _____ neurotransmitter in the G.I. tract and mediates relaxation of both circular and longitudinal muscle layers. NO serves as a neurotransmitter in areas of the brain that are specialized in _____ NO is produced in _____ during inflammation and aids them in the killing of engulfed bacteria within the lysosomes

Nitric oxide (NO) is generated in cells by the enzyme nitric oxide synthase (NOS) which metabolize arginine to citrulline with the formation of NO NO relaxes smooth muscle cells in arterioles during systole, with resultant vasodilation. NO serves as an inhibitory neurotransmitter in the G.I. tract and mediates relaxation of both circular and longitudinal muscle layers. NO serves as a neurotransmitter in areas of the brain that are specialized in cognition. NO is produced in macrophages during inflammation and aids them in the killing of engulfed bacteria within the lysosomes

Oxidative stress can result in ______ or ______

Oxidative stress can result in adaptation or oxidative damage Adaption: Mild oxidative stress can result in adaptation. In this case cells are acclimatized to elevated concentrations of free radicals because increase synthesis of antioxidants. Ischemic preconditioning is an example. Brief periods of ischemia in pig hearts lead to depression of contractile function (myocardial stunning). The contractile function returns after some hours or days. Repeated brief periods of ischemia lead to quicker return of contractile function. This is due to increased synthesis of heart antioxidants

Sources of free radicals: Peroxisomes are membrane-enclosed organelles that contain enzymes involved in a variety of oxidative reactions. A variety of substrates are broken down by such oxidative reactions including ____ acids, ____ acids and _____ acid. One of the byproducts of these reactions is _____ To remove hydrogen peroxide produced peroxisomes possess an appreciable amount of _____

Peroxisomes are membrane-enclosed organelles that contain enzymes involved in a variety of oxidative reactions. A variety of substrates are broken down by such oxidative reactions including fatty acids, amino acids and uric acid. One of the byproducts of these reactions is hydrogen peroxide. To remove hydrogen peroxide produced peroxisomes possess an appreciable amount of catalase.

Peroxynitrite (formed from NO + O2`) can cause damage to ____ and ____ Peroxynitrite can also react with proteins converting them to ______

Peroxynitrite (formed from NO + O2`) can cause damage to lipids (causing lipid peroxidation) and DNA (causing DNA fragmentation) Peroxynitrite can also react with proteins converting them to 3-nitrotyrosine

Sources of free radicals: Phagocytosis that takes place in ____ and ____ produces large amounts of free radicals. _____ are produced by a controlled reaction and are used to kill the phagocytised bacteria.

Phagocytosis that takes place in neutrophils and macrophages produces large amounts of free radicals. Bursts of free radicals are produced by a controlled reaction and are used to kill the phagocytised bacteria.

Smokers excrete 50% more of 8-oxoG in (blood or urine) than nonsmokers

Smokers excrete 50% more of 8-oxoG in urine than nonsmokers

Superoxide dismutase (SOD) is a class of enzymes that catalyze the dismutation of superoxide into _____ and _____ SOD is a _____protein

Superoxide dismutase (SOD) is a class of enzymes that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide SOD is a metalloprotein

Superoxide radical (O2`) is considered a _____ ROS --> it is mainly produced in the _____ by _____ in the ETC leaking

Superoxide radical (O2`) is considered a primary ROS (can interact with other molecules to generate 'secondary' ROS) --> it is mainly produced in the mitochondria by electrons in the ETC leaking

Superoxide radical generates other 'secondary' radical species: • Superoxide reacts with superoxide to form _____. • Superoxide reacts with _____ to form hydrogen peroxide. • Superoxide reacts with hydrogen peroxide generating _____

Superoxide radical generates other 'secondary' radical species: • Superoxide reacts with superoxide to form hydrogen peroxide. • Superoxide reacts with ambient water to form hydrogen peroxide. • Superoxide reacts with hydrogen peroxide generating hydroxyl radical

The lipid peroxyl radical reacts with other polyunsaturated fatty acids, abstracting an electron and producing a ____ Lipid hydroperoxide is unstable and its fragmentation yields small molecules such as ____ and ____

The lipid peroxyl radical reacts with other polyunsaturated fatty acids, abstracting an electron and producing a lipid hydroperoxide (CH-OOH) Lipid hydroperoxide is unstable and its fragmentation yields small molecules such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE).

There are three types of NOS: _____ is expressed in neurons. _____ is expressed in endothelial cells. _____ is induced in macrophages

There are three types of NOS: Neuronal NOS (nNOS) is expressed in neurons. Endothelial NOS (eNOS) is expressed in endothelial cells. Inducible NOS (iNOS) is induced in macrophages - note: iNOS is not expressed constitutively as nNOS and eNOS are

Three forms of SOD are present in cells: SOD1 and SOD3 contain prosthetic groups such as ____ and ____. SOD2 has _____. SOD1 is located in the _____, SOD2 is _____, SOD3 is _____ Mutations in the gene encoding SOD___ have been linked to familial amyotrophic lateral sclerosis (ALS), a neurodegenerative disease

Three forms of SOD are present in cells: SOD1 and SOD3 contain prosthetic groups such as copper and zinc. SOD2 has manganese. SOD1 is located in the cytoplasm, SOD2 is mitochondria, SOD3 is extracellular. Mutations in the gene encoding SOD1 have been linked to familial amyotrophic lateral sclerosis (ALS), a neurodegenerative disease

We already know the Fenton reaction will produce Hydroxyl radical (OH˙) --> describe how UV radiation and ionizing radiation will produce Hydroxyl radical (OH˙)

UV radiation: UV radiation in the skin splits H2O2 producing (2) hydroxyl radical --> which causes skin aging (wrinkles) Ionizing radiation (stronger than UV radiation): It splits water molecules producing hydroxyl radical, OH˙

Vitamin C is a good protection against ____ and ____ Also is a good inhibitor of _____

Vitamin C is a good protection against ROS and RNS --> it scavenges superoxide, hydroxyl radical and peroxynitrite Also is a good inhibitor of lipid peroxidation

Vitamin ____ is the most powerful inhibitor of lipid peroxidation

Vitamin E is the most powerful inhibitor of lipid peroxidation

Describe what occurs to O2 when it gets: - 4 electrons - 3 electrons - 2 electrons - 1 electron

When O2 gets 4 electrons --> it becomes H2O When O2 gets 3 electrons --> it becomes Hydroxyl radical (OH˙) When O2 gets 2 electrons --> it becomes Hydrogen peroxide (H2O2) When O2 gets 1 electron --> it becomes Superoxide radical (O2˙)

When superoxide reacts with hydrogen peroxide generating hydroxyl radical it is known as the _____ reaction

When superoxide reacts with hydrogen peroxide generating hydroxyl radical it is known as the Haber-Weiss reaction