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Topic 12.docx

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University of Waterloo
HLTH 340
Steve Mc Coll

Topic 12: Toxicodynamic Mechanisms - Protein Targets/Interference with Protein Functions Electrophilic xenobiotics & reactive metabolites  Explain how reactive organic chemicals can be classified by electron affinity. o The deal is that there are 2 major classifications to be made on the basis of electron affinity -- either you have a HIGH affinity for electrons (and are thus "electrophilic") or you are electron-rich already and so you do NOT seek out electrons (i.e. "nucleophilic" because you are seeking an electron-deficient nucleus which you can join with)  Electrophiles generally have a localized partial positive charge, and so by the laws of attraction they are attracted towards electron-rich groups  Conversely, nucleophiles have these electron-rich regions, and so they are an attractive "target" for electrophiles  Discuss some of the functional groups known for their electrophilicity. o Epoxides: the oxygen is stealing electrons away from the carbons, and so they are very electrophilic o Also aldehydes are very reactive  i.e. We know that formaldehyde is used a lot in histology/anatomy labs…or also as a disinfectant  They are potentially toxic -- they can cause allergies, hypersensitivity reactions, etc…even certain cancers in the upper respiratory tract o Also quinones are electrophilic, as are the quinonamines - these guys are a type of group formed by the metabolic phase 1 metabolism of acetaminophen  Thus while we say that acetominophen is a relatively safe drug, it is also sometimes dangerous o Also acyl dienes, haloalkyls, etc. we will discuss later  Discuss some nucleophiles which are of interest to us. o Many macromolecules and biomolecules are nucleophilic, and thus vulnerable to attack from the electrophiles  These things include the nucleotide bases in DNA  And also the --SH (thiol) groups in amino acids such as cysteine  When we put electrophiles and nucleophiles together, what happens in general? o A reaction occurs very fast (seconds or minutes), particularly when they are strong electrophiles/nucleophiles o Remember that a nucleophile is EXACTLY what an electrophile is looking for, and vice versa  Now talking specifically, what are the kinds of results which can come from an electrophilic/nucleophilic reaction? o Well the overall idea is that ADDUCTS are formed -- the result of a covalent bond between the electrophiles and the nucleophile o However, within this there are different kinds of adducts which can be formed:  One result is alkylation: the addition of small methyl or ethyl groups  Notably, this alters FUNCTION but NOT structure  The idea is that the electrophile only adds a small part of itself to the nucleophile -- often just a methyl or ethyl group  Frequently this happens to DNA, and it leads to mutations  Another result is BULKY adduct formation, which is similar to alkylation except it involves a larger molecule -- often the ENTIRE electrophile  Here we not only alter function but also STRUCTURE -- like the secondary/tertiary structure of an amino acid chain, for example  Of course BPDE is a bulky adduct that we have already discussed -- recall how the epoxide group in the BPDE will attack the DNA, forming a covalent bond…and then the bulky adduct will push itself around in the DNA and displace the normal bases in the helix  The last type is cross-linking adducts, when a covalent attachment is created between two nucleophiles, via the electrophile  The way this works is that the electrophilic group is bi- functional, meaning that it has 2 reactive functional groups in it - - so when we have this, typically one of the reactive groups will react with one part of a protein or nucleic acid and the second one will react with another  And so now there are 2 adducts formed in the DNA molecule (for example) and they are joined by the remainder of that electrophilic material, we get something called cross-linking  i.e. in the case of DNA we know we have a double helix -- but those strands are only held together by weak hydrogen bonds and so if we have a cross link through a bi-functional alkylating agent that joins one strand to its complementary strand through its universal covalent linkage, we are in big trouble b/c the strands can no longer separate during transcription of mRNA, or in DNA replication, etc. DNA bases (guanine) as a target nucleophile for electrophilic attack and adduct formation  Discuss the nature of DNA bases, and explain why they are especially to electrophilic attack and adduct formation. o Well if we imagine a helix, we see that we have the nucleotides composed of a nitrogenous base attached to a sugar-phosphate backbone  The complementary strands are linked together through hydrogen bonds between the nitrogenous bases  Each nitrogenous base is either composed of 1 ring (pyridimine) or 2 rings (purine)  The rings have nitrogens on them o The point is that the nitrogenous rings are electron rich -- they have nitrogens with free electron lone pairs, and they also have double bonds  In particular they are more electron-rich than the sugar-phosphate backbone, and so the rings are where the electrophilic attack will occur o And so the attacks occur, and they add to the rings such that the hydrogen bonds between the nitrogenous bases can no longer occur  Notably this happens to guanine often, because its electrons are the most accessible After these attacks occur, what is the result? What do we end up seeing?  o One result is that the base-pairing rules are no longer followed since the electron structure of the bases is altered so much (lots of double bonds switched, protons added, etc.)  i.e. if you have a methylated guanine, it won't pair with C anymore -- instead it will pair with T…and this will lead to a "mis-sense" mutation o Alternately, these attacks can make the entire base chemically unstable -- sometimes it falls out of the helix (i.e. we get the removal of the entire purine base) -- and this is called de-purination  And when this happens, the sugar-phosphate backbone is left on its own and it is essentially just a "ribose moiety" -- we say that the location is apurinic o Notably, all the same things can happen with pyridimines (i.e. apyridiminic site), but it is just less frequent  This is another kind of missesne, it will screw up the base pairing rules -- we will either have mis-pairing or no pairing at all in the case of apurinic sites Target and non-target nucleophiles  Given all that we now know about electrophilic/nucleophilic reactions, discuss some of the nucleophiles we frequently see as electrophile targets in the body. o Basically, they are the things which contain electron-rich groups: o sulfhydryl( -SH), amine( -NH2), hydroxyl (-OH) groups o These include certain macromolecules: o Heterocyclic structures of DNA: their nucleotide bases (purine, pyrimidines) have electron-rich regions o Also, we have proteins: the main protein targets are sulfhydryl- containing (-SH) cysteine side chains in proteins  also -NH2 (lysine and histidine) and -OH (tyrosine) rich proteins  On the other hand, what nucleophiles do we have who are "non-target" nucleophiles -- that is, we don't mind that they get attacked? Discuss one in particular. o The big one for us is glutathione (GSH), which has an --SH group that attracts electrophiles to it and reduces them (i.e. donates its electrons)  The damage of glutathione does not harm us and in fact is beneficial because it prevents the electrophiles from attacking victims of consequence o Here are a few thoughts on glutathione:  It is SO nucleophilic that it can participate in both enzymatic and non- enzymatic detoxification reactions (that is, it does not necessarily need an enzyme to help it to react)  However it also participates in enzymatic reactions: the enzyme will take the GSH and combine it with a powerful toxic agent such as BPDE in order to detoxify it  GSH is not reusable, because once it covalently bonds to a toxin, its ability to detoxify is removed -- and so we think of their supply in the cell as a finite reservoir  Thus, the amount of GSH we have is important, especially in times of drug overdose where the influx of epoxides (or other electrophiles) is greater than normal  Talk about where in the body we see GSH. o Well firstly, the liver cells have hundreds of millions of GSH molecules in the liver cell b/c this is where it is most badly needed -- recall that the liver is the detoxification center for the body  Conveniently, the liver is a major site of synthesis of GSH as well o Other tissues needing GSH in high quantities -- such as the LUNG -- receive their GSH from the liver  The lung is another high-requirement area of the body for GSH because they have to detoxify substances in the air we breathe Glutathione (GSH) as a key cellular defense against electrophiles and oxidants  Comment on the chemical structure of glutathione, and the implications of this for its depletion and restoration. o We see that it is a glycine and glutamate molecule linked together by a cysteine (i.e. it is in the middle)  The thing is that the cysteine in the middle has an SH (thiol) group, and as you may recall, that is the group we care about because it reacts with the electrophile o But the thing is, sometimes GSH is OXIDIZED -- this means NOT that an oxygen is added, but rather that a hydrogen is removed -- and in fact the hydrogen on the thiol group is removed  And when this happens, the remaining sulfur joins with a sulfur from ANOTHER GSH molecule, and a glutathione dimer is formed o This is actually a bad thing, because the functionality of the thiol group is removed and so GSH is ineffective o One implication for this, then is that when we say that GSH levels are depleted in a cell it doesn't always mean that it disappeared -- maybe instead it just lost its capacity to do stuff since it was oxidized  Thus it is somewhat good news, because we can fix this by adding hydrgoens to reduce the molecule again so we get a working form of it -- these reactions are known as redox reactions  So these things are very important not only b/c they help the cell to defend itself against those epoxides but also because they balance reducing and oxidizing equivalents within a cell  Notably a healthy cell will have a bit more reductants than oxidants, and it is largely through glutathione that we have this reserve of reductants that keep the cell healthy Direct-acting and indirect-acting electrophilic agents  (A bit of a review, but) explain the major difference between direct-acting and indirect- acting agents. o Direct-acting agents are those which can "immediately" react with a substance because the parent chemical -- i.e. the initial form -- is chemically reactive  Thus it does not require metabolic bioactivation by P450 or other enzymes o Conversely, indirect-acting agents are not initially reactive and so they require that activation step  Alright, more on direct-acting agents now. Discuss some of their pertinent properties. o They often contain reactive electrophilic group in molecular structure  They are either monofunctional = single reactive group...  …or bifunctional = 2 reactive groups o They are usually short-lived in environment (minutes, hours) because they are so reactive  The reactions are
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