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Topic 14.docx

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HLTH 340
Steve Mc Coll

Topic 14: Toxicodynamic Mechanisms - Free-Radical Toxicity Oxidative Stress and Free Radical Toxicity  What are some characteristics of free radicals? o They are reactive molecular fragments with a SINGLE UNPAIRED ELECTRONS  This happens because sometimes a covalent bond (2 electrons) is broken such that one electron goes to each molecule o So the fragments with single electrons are very chemically reactive because to have one electron is to be unstable - so they want to react with anything that will change this situation for them o Lastly, we note that the free radicals often attack important cellular macromolecules (more later)  Talk about some of the cellular targets for the free radicals. o Their primary target is the phospholipids that make up the various membranes in the cell (the outer membrane, the mitochondrial membrane, even the nuclear membrane)  Specifically the lipid tail will be attacked in a process called "lipid peroxidation" o Proteins and DNA can also be attacked  This results in genotoxicity, mutation, cancer, etc.  Explain the notion of oxidative stress in general. o It is the amount of oxidation reactions which occur in the cells due to free radicals which also interact with oxygen o As mentioned before, the macromolecules of the cell are frequently targets of these reactions, and so when free radicals come and produce oxidative stress, often the macromolecules bear the brunt of the damage o Notably, it is not only free radicals which cause damaging oxidative reactions in the cell:  For example, we can have oxidative phosphorylation -- it is a natural physiological process which makes ATP…but this is a very oxidative series of reactions…  Mitochondria even more so than the cell membrane is subjected to a lot of oxidative stressors including free radicals -- when they undertake the stuff they get out of control and it causes mitochondrial damage  Also the smooth ER is a source of problem -- remember that all the p450 are strongly oxidative enzymes so the ser also has a lot of oxidative enzyme reactions taking place -- it too can be damaged b/c it is also made up of phospholipids  All in all, typically membrane systems in the cell can be damaged b/c they are made up of phospholipids but also they contain p450 (in the ser), oxidative phosphorylation enzymes (mitochondria), and so on Biological Effects of Oxidative Stress What are reactive oxygen species, and where do they come from?  o ROS are the larger class of damaging molecules which include free radicals  Notably, oxygen can also be converted to single-electron molecules (through normal metabolic processes) and thus become reactive oxygen species o There are 2 main categories of ROS sources:  exogenous ROS sources (environment)  So for example, we are talking about air pollution  Stuff like smog etc is oxidative air pollution -- if we looked at the components we would say it is strongly oxidative -- it will cause crap in the lungs, eyes, skin, heart even, etc.  Some of the things we breathe in are either free radicals or can give rise to free radical reactions…  endogenous ROS sources (internal)  Remember that we can MAKE ROS in natural reactions as well - - and so if the body undergoes some sort of regulatory abnormalities…it can upset the balance of stuff…it can sometimes give rise to oxidative stress and free radical reduction not so much by chemical means but more regulatory means  Example: some of the endocrine disruptor chemicals like pcb/dioxins aren't oxidative themselves but they can perturb the cells to give rise to oxidative stress, perhaps by inducing the production of enzymes involved in oxidation  What is a redox state, what is its significance to the cell, and how does the cell attempt to interact with it? o Redox state is just the state of the cell with respect to oxidants or reductants -- if there a lot of oxidants, then we will tend to see oxidation reactions happening and if there are a lot of reductants we will see more reduction reactions happening o It is important to the cell because remember -- although oxidation reactions are occasionally useful, they cause damage and we don't want them to happen too much  So when we have reductants, they can neutralize the undesirable effects of the oxidation o Thus the cell uses various homeostatic mechanisms and tries to maintain a state of reductants being slightly surplus over the oxidants  How does the cell maintain its optimum redox state?  Cells try to maintain optimum redox state by creating and storing reducing equivalents  i.e. NADPH, GSH, vitamin C, etc  Basically, these guys serve as reserves of antioxidants  What happens when we lose "redox control", i.e. it gets out of hand and we get too many oxidative substances?  the cell undergoes relatively uncontrolled reactions which will cause damage to dna, protein, cell membrane, etc. through phospholipid damage  These can be "dominoed" as well, whereby damage in one area like phospholipid damage may in turn increase dna damage  Other times cells have sensors that can sens oxidative stress -- we know that the nrf2 and the keap system will kick into action and up-regulate a lot of the phase 2 enzymes, antioxidants, etc.  Notably however, this is itself a stressor b/c the cell has to deplete its resources to manufacture this material  If it is really uncontrollable we will get an accumulation of damage to key target molecules…we will also get the molecule breaking down chemically…and when this happens it can give rise to toxic by products  Notably, we see that normally the initial attack is bad but the secondary chemical reactions "downstream" can be as bad or worse  So this is why we say that oxidative stress is not just about free radicals but also about all the different particles and pieces that can be created as a result -- so it screws up metabolism, regulation, organizational structure of the cell, etc.  So a few things can happen as a result:  Unregulated cell death (bad because all the stuff will come out) or apoptosis (programmed cell death which is "cleaner" -- this often happens during oxidative stress -- so oxidative stress can kill the cell outright or cause the cell to be damaged so much that it does programmed cell death  However, other times the cells recover -- they fight off the attack and get back to normal…so this is possible…  Relate this discussion back to aging.  We get a burden of oxidative byproducts in cells/tissues over decades -- so many of the theories of why our tissues age are related to this free radical damage  The theory is that we can never totally eliminiate it -- as we get older, the byproducts form this stuff build up in the cell like a sludge, and these byproducts are either themselves toxic or they represent a byproduct of toxicity  So if we were to take a small sample of heart tissue from us…the cells would be pretty clean…but if they took it from 58 year old heart…it would have a large quantity of a substance called lipofuscin -- this is seen as an indicator of accumulated oxidative damage  So by the time we are old like 58, 2 or 3% of all the dry weight material in the heart cell is made up of lipofuscin…and it is simply this sort of accumulated sludge that builds up as a byproduct of years and decades of oxidative stress  So whether the lipofuscin is toxic or not is debatable…but it is a sign that we get a lot of crud built up -- lipo means that it is lipid rich -- breakdown of natural cellular phospholipids…and fuscin means that it glows purple/pink after you put a dye on it…  Describe a flow diagram that summarizes this process neatly. o [See slides, it is important]  Talk about NADPH, and explain its relevance to this discussion. o Recall that Vitamin C and GSH help increase the reduction part of the cell because they can neutralize oxidants, but by far the BEST reductant is NADPH o Recall that it is a molecule that is created by metabolism of fats and sugars and they are strongly reducing because the hydrogen that this molecule carries has 2 electrons o So the main cell fuel for reducing power in the cell is nadph and that's why keeping the cell active in terms of metabolism is important -- you have to keep making nadph through glycolysis or oxidative phoshphorylation in order to ensure that we have a good supply of the material o NADPH is also important because it is linked to the other defense systems, which is why we have that "altered repair and defense capability" box on the flow chart:  For example, glutathione reductase is an NADPH dependent system…so nadph will feed reducing equivalents into glutathione -- glutathione then feeds it into other defense systems like vitamin C  so there is a system of interrelated reductive systems in every healthy cell that keeps it in a reduced state…so it is when we lose energy and have an electrophilic or free radical attack and we deplete our glutathione, the cell loses its redox status and we have a problem  Given the discussion on NADPH, how do anti-oxidants fit into the picture? What cautions should we take with them? o Antioxidants are valuable because they can neutralize electrophiles/oxidants and thus maintain our store of NADPH at an acceptable level  Thus we normally have is a ton of antioxidants that we keep in reserve o Given this knowledge, a lot of studies have been done as to whether we can slow down cell damage, prevent aging, etc…reduce the likelihood of cancer…if we use antioxidants -- b/c if they fight the stressors caused by oxidative stress, then increasing the amount of antioxidants we take into our bodies is a good thing right? Well maybe but may not:  What has been found is that if we have a healthy diet, it seems to be protective - -a veggie and fruit rich diet show conclusively that it protects against certain kinds of cancer, other oxidative stress mechanisms, increase likelihood of cardio diseases down, etc.  HOWEVER -- the concept of chemoprevention (i.e. oltipraz, where we increase phase 2 enzymes to prevent liver cancer due to aflatoxins) turn out to be bad ideas -- some studies just take an antioxidant and apply one or a few of these antioxidant substances to people in high doses -- and what has been found is that sometimes the substances are protective -- but other times they INCREASE the rate of cvd etc  The point is that you can't just give a person one big dose of this stuff and expect it to work well b/c the antioxidant system within the cells and tissue is an interlinked system -- just dumping a lot of Vitamin C is not likely to help if we have inadequate stores of glutathione for example because we need that glutathione to keep the vitamin C in a reduced situation -- in fact, it will create an imbalance and make things worse Exogenous and endogenous sources of oxidative stress  What are some exogenous sources of oxidative stress? o Photochemical air pollutants -- because sunshine is needed to produce these guys via reaction (think smog)  NO2  SO2  O3 o Pesticides  Paraquat (herbicide) -- thought to be involved with Parkinson's  Vacor (rodenticide) o Foods  Sodium nitrite (food preservative) -- often used with ham, bacon, etc.  Fava beans o Drugs  Sulfonamides (antimicrobials) Chloroquinones (antimalarials)   Bleomycin (antitumor)  Alloxan (insulin synthesis inhibitor) o Chemicals  Naphthalene (mothballs)  Trinitrotoluene (TNT, an explosive) -- nitro derivatives are dangerous  List some endogenous sources of oxidative stress, and comment where necessary. o Reactive Oxygen Species .  Superoxide radical (O 2 -- these can be created by physiological reactions OR the reaction of xenobiotics  Hydrogen peroxide (H2O2) -- it is a direct oxidant (?)  It is often used as a disinfectant or to bleach hair  Hydroxyl radical (OH .) -- it is the most reactive -- there is no way to prevent it from reacting with DNA or protein o Organic hydroperoxides  Lipid hydroperoxides (LOOH) -- remember that this is what happens as a result of lipid peroxidation  Other hydroperoxides (ROOH) o Bioactivated free radicals (substances which have been bioactivated into a radical form)  Carbon tetrachloride: it is not harmless, but the P450 system in the liver ACTIVATES it into a radical form that is very toxic -- it is hepatotoxic  BaP radicals (carcinogenic as we know) -- it can become BaP quinones, which are bad The BIG message to note here is that both electrophilic (previously  discussed) and free radical (here discussed) species can be bad for the body Photochemical Air Pollution  Where do we see smog, and how is it created? o First we will discuss how it is created, because then its presence is easier to predict and understand: it is created by action of sunlight (UV radiation) on automotive exhaust and other combustion emissions (industrial, power generation, etc.) o Thus we mostly see it in urban areas and downwind regions  Urban areas: because this is where we have a lot of car exhaust, and even power-generation plants to some degree  Also, we see it in downwind regions because this stuff is a gas and can be blow to different places by wind currents  For example, we get smog from American midwestern states  Talk more specifically about the constituents of smog. o It is a pro-oxidant mixture of toxic gases and particulates  "Pro-oxidant" means that it causes oxidation reactions to happen in the body (remember we don't like this)  The toxic gases are a mix of primary and secondary pollutants:  Primary pollutants (i.e. sulfur oxides) are already in the car exhaust, power plant emission, etc.  Secondary pollutants are the RESULTS of chemical reactions in the air (c
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