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Topic 8.docx

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University of Waterloo
HLTH 340
Steve Mc Coll

Topic 8: Toxicokinetics - Metabolism Part 1 Xenobiotic Metabolism - biotransformation of lipophilic compounds  Give a very brief and general overview of what we mean when we talk about xenobiotic metabolism. o OK firstly, realize that we are NOT talking about metabolism in its more common sense: glycolysis, Krebs Cycle, and so on -- instead, we are talking about the series of reactions which a xenobiotic experiences upon entering the body o Notably, the term "biotransformation" is synonymous with metabolism (at least for our purposes)  Give a very high-level explanation of how we can characterize xenobiotic metabolism (in terms of phases). o We are saying that we can consider xenobiotic metabolism to happen in two phases: Phase 1 and Phase 2  Phase 1 involves the degradation of the xenobiotic: we are breaking down the parent compound to other products  Notably, these other products may be less reactive/dangerous or MORE reactive/dangerous: the body doesn't "know" and therefore cannot act judiciously  While Phase 2 involves the conjugation of the xenobiotic: we are adding a small endobiotic (i.e. made within our body) molecule to the xenobiotic molecule  Talk about the NATURE of most Phase 1 reactions. What are the 4 big characteristics? o Most Phase 1 reactions are oxidation reactions, catalyzed by enzymes from the "mixed function oxidase" family (which we refer to as MFO or P450)  The "P450" is because the MFO's have a cytochrome (colored enzyme) component which has a heme group, whose color wavelength is ostensibly 450 nm (red)  This cytochrome component has a heme group, which o Although we remember that oxidation reactions can mean different things (i.e. loss of H, loss of e, etc.), in this case oxidation means the addition of a SINGLE OXYGEN ATOM (from O2) to the xenobiotic  Because of this, the MFO/P450 are also known as mono-oxygenases o We also note that often there are MANY metabolites resulting from these Phase 1 oxidation reactions (thus it is not so simple as to say a xenobiotic always leads to the same metabolite) o Lastly, these oxidation reactions often result in "functionalization": the bestowing of a new functional group on the xenobiotic  This functional group gives the molecule distinct properties that allow it to participate in a certain class of reactions (i.e. the Phase 2 reactions)  Unsurprisingly, most of the functional groups contain oxygen  What kinds of xenobiotics often undergo this Phase 1 metabolism? What issues come out of this? o We see that lipophiles are preferentially oxidized in Phase-1 metabolism -- we add functional groups to them and make them hydrophilic  One result of this is good: since they are hydrophilic, they are now less persistent because they can't dissolve in our adipose tissue cells and ALSO they are now MORE EASILY excreted  This is necessary for our own lipophilic hormones, too: we use Phase 2-type reactions to eliminate things like estrogen so they don't build up  However, adding the functional groups makes them potentially more reactive, which in turn means that they could be more toxic  What is the goal of the Phase 2 reactions? How does Phase 1 allow this to be realized? o The purpose of this is to neutralize any damage that has been done in Phase 1 (i.e. making the xenobiotic more reactive by adding functional groups), and so these are also known as DETOXIFICATION reactions  We do this by attaching a harmless endobiotic onto the xenobiotic which will negate any toxicity that may have been "added" due to the Phase 1 reactions  It is able to negate the toxicity because almost all endobiotic molecules are ionic (i.e. it could have a carboxy or sulfate group), and so it is very hydrophilic (a good thing, for the reasons given earlier) o Ironically, the reactions we are worried about in Phase 1 (the addition of the functional group) ALLOW these Phase 2 reactions to happen because there MUST be a functional group on there in order to perform conjugation o Lastly, we will note that in a small percentage of cases, conjugation actually makes a xenobiotic MORE toxic Microsomal and Cytosolic Enzymes  Talk about the tissues in the body where we would expect to see biotransformation occurring. o Well firstly, the liver is the most active biotransformation organ -- in fact, biotransformation is its major job (recall how all ingested foods pass through the LIVER first)  The liver also stores and exports vitamins, metals, etc.  Liver cells are called HEPATOCYTES o However, the lung, kidney, other tissues also have significant activity  This makes sense: these are the tissues which are also exposed to high levels of XENO-molecules (molecules that we get from OUTSIDE)  The lungs are exposed to vapors  The lining of the intestine contacts all our food  The kidney ends up excreting our XB's  What does all of this mean in terms of WHERE we are going to see the effects of xenobiotic toxicity? o It means that often we will see the liver get pounded (i.e. cirrhosis -- scarring/damage of the liver) because if the Phase 1/2 reactions go awry and a xenobiotic is activated to become MORE toxic, the liver cells are going to be right there o However, the liver is also a PROCESSING center and so it can send the xenobiotic to other parts of the body where it will do damage  Discuss the relative locations of the Phase 1 and 2 enzymes within the average hepatocyte. o Phase-1 (P450) enzymes are always microsomal (i.e. embedded in the smooth endoplasmic reticulum)  This is because the enzymes require the phospholipid environment in order to catalyze their reactions o Phase-2 enzymes are predominantly microsomal...  These include epoxide hydrase (EH) and glucuronyl transferase (GT) o …although some are found in the cytoplasm  These include ‘soluble’ forms of glucuronyl transferase, sulfotransferase (ST), and glutathione-S-transferase (GST)  Expound yet again on the Phase 1 reactions. Talk about reactants, co-factors, and products. o Reactants are interesting because unlike most other enzymes, the MFO's can work with ANY xenobiotic -- the only requirement being that it is LIPOPHILIC  Because of this "come one come all" nature, they are often called "promiscuous" o Also, certain co-factors are necessary (co-factor meaning that these compounds are needed to make the reaction go, but are not central to it)  One of these is (obviously) oxygen: because we are OXIDIZING the xenobiotic and we will need to get oxygen from somewhere!  The other one is NADPH, which is a high-energy endobiotic molecule -- we need this because the oxidation reaction is evidently not spontaneous  Just briefly make a few points about the Phase 2 reactions. o As we know, we are just conjugating an endobiotic molecule onto the functional group provided by the first reaction  This often allows for fecal or urinary excretion o Also, note that phase 2 reactions need energy -- hence the presence of ATP/UTP on the diagram Phase-1 metabolism - mixed function oxidase (MFO)  Wow, it seems like we are talking about the same stuff over and over. Explain how Phase 1 reactions are applicable to 2 major classes of lipophilic substrates. o Well firstly, as we know, Phase 1 reactions occur to lipophilic xenobiotics  These include chemicals, drugs, and synthetic steroids o But ALSO, Phase 1 reactions apply to ENDOBIOTIC substances that are lipophilic  These include various fatty compounds (think cholesterol!) and also endogenous steroids (i.e. estrogen, as discussed earlier) o Notably, the specific P450 enzymes (remember that we usually talk about them as a general family) and the locations in the body where the reactions occur on endobiotic/xenobiotic are DIFFERENT  That is, different enzymes within the family will handle either endo or xeno  Also, in the liver we do more xenobiotics, while in the testes we would be handling the endobiotics  Discuss some of the oxygen-containing functional groups that are attached in Phase 1 reactions. o R-OH (alcohol derivatives) o R-COOH carboxylic acid derivatives: this is good because it can be deprotonated and become COO-, which is even more hydrophilic  Note that the presence of TWO oxygens when our enzymes are MONO oxygenase is because we add the oxygen first, then the thing gets "recycled" back through the enzymes and another oxygen is added o R>O epoxide derivatives: these are compounds where a "triangle" is formed between an oxygen and 2 carbons  These are crazy b/c they are very toxic (recall ring can be easily broken) -- so one important issue is, "How do we get rid of these before they do a reaction that causes damage?"  So these are TOXIC REACTIVEI NTERMEDIATE and we need to deal with them MFO - mixed-function oxidase complex  Give the full chemical equation for the typical MFO reaction. Talk specifically about the role of NADPH here. o Equation: R-H (lipophilic substrate) + O2(molecular oxygen) + NADPH + (reduced cofactor) ---MFO---> R-OH (oxidized metabolite) + H O 2 NADP (oxidized cofactor)  This is known as a "redox" reaction, because both reduction and oxidation are occurring at the same time (the lipophilic substrate is oxidized, while molecular oxygen is reduced) o We have to realize here that it is NOT a spontaneous reaction -- in fact, it is very difficult to break apart the molecular oxygen so that we can attach one of the atoms to the lipophilic substrate  Our solution for this is to use NADPH (nicotinamide adenine dinucleotide phosphate), which is a "reducing cofactor": it provides energy to drive the reaction and also provides the hydrogen (plus electrons) which we will use to reduce oxygen  The hydrogen/electron pair package is known as a REDUCING EQUIVALENT o Lastly, note that 100% oxidation happens because the equilibrium is completely to the right  Because of this, we call the MFO's "enzymatic blowtorch" because they so EFFICIENCTLY and COMPLETELY "burn up" or "oxidize" the lipophilic substrates  Given all this, consider the full name of the machinery which handles this reaction: it is the "mixed-function oxidase complex", which consists of cytochrome P450 AND NADPH/P450 reductase". Break this down and explain each part. o OK firstly, the cytochrome P450 is what we have encountered before: it does the oxidation of the lipophilic substrate -- so this is not very complicated o How
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