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Topic 9.docx

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University of Waterloo
HLTH 340
Steve Mc Coll

Topic 9: Toxicokinetics - Metabolism Part 2 Enzymatic and induction properties of the P450 isoforms  Explain what the notion of "substrate preference" is, within the family of P450 enzymes. o The idea is that P450 enzymes are one big family of enzymes which can oxidize a wide variety of lipophilic substrates (recall that they are considered to be "promiscuous") o Within this family, each isoform is slightly different in its catalytic properties and also the degree to which it is inducible (a very important feature)  What this means is that different isoforms will specialize in different types of lipophilic substrates -- so each of these guys may prefer a big or small molecule…or with certain kinds of benzene or no benzene rings in it…  The other consequence is if one enzyme is missing or deficient, another could do the job (although not as well)  Talk more about the implications of that last statement -- that 2 or more P450 isoforms may metabolize a given xenobiotic. o This results in the notion of primary and secondary metabolic pathways: the idea that 2 or more P450 isoforms are able to do whatever -- but one of them could do it more quickly and so it would be designated as primary  Then the other isoforms that have a lower affinity for that particular XB would be considered secondary metabolic pathways because the rate of metabolism by these less preferred enzyme systems is SLOWER  When we are looking at this system from the outside, why is it hard to predict exactly how these pathways will work? o It is because the pathways are unpredictable due to genetic and environmental factors (alcohol, smoking, etc.)  As for genetics, we can have polymorphisms that will give us more or less of certain enzymes  And the environment can also affect the systems, making them more or less active  For each enzyme, besides the substrate type which it works with the most easily, what else is a unique and important property of these enzymes? What is the result of this? o It is their maximum capacity: the fastest rate at which they can catalyze the reactions  This capacity is defined by the number of catalytic binding sites they have o This limitation results in dose-dependent kinetics, which is the idea that the size of the xenobiotic dose will affect how fast it gets processed  When the dose is low, the catalytic binding sites are not overcrowded so the enzymes can work at maximum speed  However when the dose is higher, there will be a "line up" and so the reactions will go more slowly -- we say that the enzymes have been "saturated"  How do all of these traits result in "low" and "high" capacity pathways? o We get alternate metabolic pathways that are either low or high capacity because each pathway will include certain types of enzymes that will have different capacities than others  Even more so, different TISSUES in the body will have different NUMBERS of different enzymes o This all adds up to different tissues having different pathways that are either low or high capacity (due to the nature of the enzymes constituting the pathway and also the number of enzymes there are) o Of course we should note that all of this is subject to genetic and environmental factors  What are the 3 hierarchical levels of biochemistry study that are relevant to this? o Firstly, as we discussed, we all have different genetic makeup -- and this affects the enzymes -- so "genomics" is important o But on a "closer" level than that, we also have proteomics  i.e. the genes themselves could have certain properties -- but more important is what comes out of them b/c this is what actually makes effects o But the highest level is metabolomics -- doesn't matter what proteins and enzyme we have, it is what the cells do with them in the various pathways Regioselectivity - alternative metabolic pathways  OK, we have already discussed a situation (last topic) where we have alternate metabolic pathways run by different enzymes which result in different functional groups being added/removed from the molecule. Now explain another case where REGIOSELECTIVITY makes the difference…and give a general overview. o OK, regioselectivity means that depending on the conditions/environment, the functional group will be added to the molecule in a different place -- so it's not that we are adding a different group, it's that we are adding the same group but in a different place -- we are forming POSITIONAL ISOMERS  The other crazy thing is that unlike past alternate metabolic pathway situations we have studied, the enzymes for each pathway are very closely related -- it is just different isoforms of P450 o The xenobiotic in question here is bromobenzene (you know what this looks like), which is a GASEOUS substance which (like malathion) is not inherently toxic but can be BIOACTIVATED into a toxic form  And as we might expect, the pathway it takes determines whether it becomes toxic or non-toxic o We see that bromobenzene often affects the lung or the liver -- specifically it hits the organelles (such as DNA) because remember it is a xenobiotic which freely diffuses into cells because it is LIPOPHILIC, then gets activated after entry into a toxic substance  Talk about how the pathways themselves actually work. o Firstly, the TOXIC pathway is activated by the "2E1" isoform of P450, and it oxidizes the 3 and 4 carbons in the benzene ring, creating an epoxide  Thus we get bromobenzene 3,4-epoxide, which is very toxic and will cause liver necrosis  It is so reactive that it will slowly rearrange itself into para- hydroxybromobenzene, which is water soluble and can be excreted in the urine (thus the harmful substances do not last forever) o The non-toxic pathway is run by the "1A2" isoform of P450, and the 2,3 carbons get made into an epoxide, which is not as bad  This can also self-rearrange SLOWLY to form ortho- hydroxybromobenzene, which is again soluble and can be excreted in the urine  Why is one positional isomer (3,4 epoxide) toxic, and the other one (2,3) non-toxic? o It is all about the reactivity of the compound -- epoxides are normally very reactive because the oxygen is much more electronegative than the 2 carbons it is bound to, so it grabs charge from them and becomes extremely negative, while the carbons are positive o However, in the non-toxic arrangement, the bromine is also close by and it is also electronegative, meaning that it can compete with the oxygen for the charges on those carbons and lessen the imbalance  How can environmental factors affect this particular proportion of pathways (i.e. influence one pathway over the other)? o ALCOHOL is a big thing: a secondary pathway for alcohol metabolism involves the 2E1 enzyme and so if we drink a LOT of it, then the 2E1 will have to get up- regulated  This is BAD because now when bromobenzene (or whatever else) comes in, the 2E1 will be more likely to metabolize it and so we get bad effects o On the other hand, SMOKING is protective in this case because the carbon in the smoke is broken down by the 1A2 isoform, and so if that upregulates then we will have more of it Enzyme inhibition and enzyme induction of the P450 isoforms  What is enzyme inhibition in general? Describe how it happens with the P450 system in particular. o Inhibition means that in some instances, the activity of an enzyme can be reduced by interfering with its catalytic activity in different ways o With P450 (and in general), the two main types of inhibition are "reversible" and "irreversible":  Reversible inhibition is "saturation competition", when one substrate overloads the binding sites on the P450 system, preventing the substrate we want from getting on there  It is reversible because if the undesired substrates leave, the good ones can hop on  Irreversible inhibition is when we attack the enzyme with chemicals and drugs and wreck it permanently  What is one example of reversible inhibition we discussed in class? o The issue is that methyl alcohol is a form of alcohol that is metabolized by the P450 system into formaldehyde, which is very dangerous (more later)  Note that this is an example of a bioactivation reaction producing something toxic from something harmless o Thus during cases where excessive methyl alcohol has been ingested, it is treated by administering 10:1 ratio (or so) of ETHYL alcohol, which is the type found in regular beer and is metabolized by the SAME system into acetaldehyde (not harmful) o Thus with ethanol occupying all the active sites of the P450 enzymes, methyl alcohol remains unmetabolized and therefore unharmful  Eventually it is breathed out of the body  Comment on the various substances involved in the previous reaction. o Formaldehyde (metabolite of methanol) is dangerous: it will attack the optic (visual) nerve -- so one of the first symptoms of methanol poisoning is the person going blind or they have severe visual impairment  Or they can infect other parts of the brain and kill you o Ethanol is often available in hospitals to be administered via intravenous drip, because of this very concern/use o Acetic acid or acetaldehyde is the raw material for the CAC so it actually gives us energy -- much better than formaldehyde effects  Explain how irreversible inhibition might work. Why does this have an application in the lab? o We may have a certain compound (a "suicide substrate") which is partially oxidized by the P450, but then it binds to the catalytic center and never leaves, thereby inactivating the enzyme and also making itself useless (hence "suicide")  Since it never leaves, we classify this as irreversible o The application is that different substrates will do this to different types of P450 systems, and so by introducing different ones and then seeing which P450 systems die, we can identify which ones are present  Give an overview of the concept of enzyme induction. o The idea is that for some reason (more later), we get increased levels of a p450 enzyme -- or more specifically a certain form or type of P450 enzyme -- in a tissue o The two reasons for this are:  Increased enzyme synthesis (common) -- perhaps due to environmental factors  Reduced enzyme degradation (rare) o The various factors affecting this process result in each person having a characteristic amount of different kinds of enzymes, and this is known as his or her "enzyme profile"  Talk about the concept of "inducers", and how this leads to up- and down-regulation. o Inducers are substrates for the enzyme that when present in greater quantities, compel the body to make more of the enzyme (this is "up-regulation") o Whereas when they are present in lower quantities, the body understandably makes fewer enzymes ("down regulation") o Each P450 isoform has its own specific xenobiotic inhibitors and inducers -- did you note the significance of that? Xenobiotic means it comes from the OUTSIDE, which means that outward factors can affect our inner enzyme profiles  One related field which is beginning to grow popular is how there are many naturally occurring substances in our FOOD that are not vitamins or nutrients, but they are important in our diet b/c they are modulators/regulators of XB metabolism  So if we consume grapes, oranges, etc…grapefruits etc…cabbages/brussel sprouts…all of these things are rich in different types of alkaloids -- non nutritive plant constituents -- and these in turn inhibit certain enzyme systems and induce others…so we are realizing that one of the reasons why everyone's enzyme profile is so dynamic is because we EAT DIFFERENTLY Cytochrome P450 (CYP) genetic superfamily  Explain what this superfamily is. o It is the P450 gene superfamily, which just means that it is the collection for all the different "isoforms" or "versions" of the P450 enzymes (there are 50 or 60)  Remember that we have different ones for different substrates, and that they can differ in terms of how they do their oxidation, regioselectivity, what kind of substances induce/inhibit them, etc.  However of course they always share the classic P450 traits: oxidative capacity, embedded in lipid membrane, require NADPH P450 reductase to supply reducing power, etc. o Nomenclature works in a "family"-"subfamily"-"individual member" format, whereby something like 3A4 would belong to the CYP3 (cytochrome P450) family, subfamily "A", and member 4 o It is a GENETIC family, thus they are classified based on base sequence -- this is better than basing it on their substrates because substrates overlap amongst enzymes o There are just 5 or 6 CYP isoforms that are the MOST COMMON, and we note that 3A4 is the #1 stunna Cytochrome P450 (CYP gene) evolutionary tree  When we look at how the P450 enzyme system evolved, what are some things we notice? o Firstly we see that while prokaryotes don't have p450, every eukaryote has SOME FORM of p450 o Also we note that the higher organisms like mammals have more (and more varied) P450 enzymes, whereas the less advanced have fewer o We notice that GENE DUPLICATIONS occur, and the duplicated genes evolve new functions and that is how the higher animals have more in number and more advanced things o We also notice the evolution in function:  First they were dealing with lipid metabolism (endobiotic)  But then moved onto stuff like cholesterol (endobiotic)  And THEN…we start to metabolize xenobiotics not only endobiotics: drug metabolism  And then animal/plant warfare started: when green plants first colonized solid ground, the ones who were selected to survive were those that could be poisonous to animals  However animals "responded" by selecting for those who had new and improved P450 s
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