Lesson 3 Flashcards
Which are the compound affected by non-microsomal oxidations?
- Amines: biotransformed by monoamino or diamino oxidases. An important one is epinephrine (important for parkinson disease)
- Alcohols: biotransformed by alcohol dehydrogenases
How can we divide oxidation reactions?
According to their localization:
- in microsomes: microsomal oxidations. CYP mediated or FMO mediated
- in cytosol or other organelles: non-microsomal oxidations
Which are the different kind of microsomal oxidations CYP mediated?
- Hydroxylation: adding of OH group on a CH3 carbon (aliphatic) or on an aromatic ring (aromatic, ex: benzene)
- Heterocycle epoxidation: formation of an epoxide (-O-) in a heterocycle. Ex: aflatoxin B1 become alfatoxin B1 epoxid
- Aromatic epoxidation: epoxidation on an aromatic ring
- N- or O- dealkylation: the alkyle is oxidated to the correspondent aldehyde (dealkylated and then linked to H or O)
- oxidative desulfuration: CYP introduce an O and the S will be released. An oxidated metabolite is formed
OPEN QUESTION: describe the cytochrome P450 catalytic cycle with NADPH-cyt450 reductase
1- Binding of a substrate to obtain the complex P450-substrate (note: the oxidation number of central Fe (iron) is +3).
2- Donation of the electron to the complex: it comes from NADPH cyt P450 reductase (note: the oxidation number of central Fe is +2, since the complex received an electron).
3- Binding of molecular oxygen to the heme Fe: the complex is now composed by O-Fe-substrate (this causes the iron oxidation number to be +3 again (= oxidized)) N.B. nothing happens to the substrate until the next step: it has not been bio-transformed yet.
4- Donation of a second electron either from NADPH cyt P450 reductase or NADH cyt b5 reductase. This causes the reduction of the molecular oxygen into the peroxy-group which is a highly unstable compound.
5- Disassociation of the peroxy-group following a double protonation: one H2O is released forming a highly reactive iron.
6- Release of the oxidized substrate (= R-OH, hydroxylated metabolite, coming again free), restoring the initial situation of Fe3+.
What do you need to recreate a cytP450-mediated reaction in vitro?
- The enzyme source: microsomal membranes
- The substrate: the drug
- Molecular oxygen O2
- NADPH: there is no need to introduce in the system also NADH. Everything works perfectly even without it.
- 2H+
Why do we call P450s ‘monoxigenases’?
starting from molecular oxygen, they introduce an oxygen atom into the substrate and they use the other to form a molecule of H2O, which is then released
Which is the specifiity of the different cytochrome P450?
They have a low substrate specificity with preferred substrate:
- CYP1A subfamily: large planar molecules
- CYP2B subfamily: molecules that targer the CNS and endogenous substrates as testosterone
- CYP2C subfamily: thrombolytic drugs as warfarin and non-steroidal anti-inflammatory drugs
- CYP2D subfamily: cardiac drugs
- CYP2E subfamily: short chain alcohols
- CYP3A subfamily: metabolization of various drugs and endogenous compounds (oxidative biotransformations)
OPEN QUESTION: how does the FMO-mediated microsomal oxidation work? Which are its substrates?
The flavin monooxygenases (FMO) usually perform N-oxidations or S-oxidations of secondary and tertiary amines.
1- Formation of a complex with NADPH+ H + in order to have a FMO-FADH2-NADP+ complex.
2- A molecular oxygen O2 is bound, forming FMO-FADHOOH-NADP+
3- The substrate X binds the enzyme, and it is rapidly released as oxidized (XO). The remaining complex is FMO-FADHOH-NADP+
4- Release of NADP+ and H2O with the restitution of the initial complex FMO-FAD.
The typical substrates are:
- Albendazole: anti-parasitic drug
- Fenbendazole: anti-parasitic drug
- Nicotine: which is N-oxidized by FMO
- Lidocaine: anesthetic
Which enzymes perform the reduction reactions?
microsomal or cytosolic reductases or oxidoreductases (dehydrogenases) with the reducing equivalents provided by either NADPH or NADH.
Also NADPH-cyt P450-reductase may be implied.
What is a semiquinone?
Semiquinone is a reactive metabolite that forms ROS, responsible for cytotoxicity in tumor cells.
OPEN QUESTION: give an example of a reduction reaction which involve NADPH cyt450 reductase
This is the case of an estrogenic mycotoxin called zearalenon (ZEA).
The reaction occurs in two senses (oxidative and reductive), but the reduction direction largely prevails on the oxidation one.
According to the species, we may have a different outcome: for pigs, the production of an isomer called α-zearalenol (OH group is below the molecule plan), for poultry, the production of β-zearalenol.
Only the α-isomer has the same properties of the parent molecule (zearalenone) and a major estrogenic activity.
α-zearalenone is more lipophilic and more prompt to reach estrogenic receptor than the parent molecule (the higher lipophilicity explains the higher affinity for estrogenic receptor).
Which are the bond affected by the hydrolytic reactions?
These reactions involve a water molecule being added:
- To ester bonds thus releasing a carboxylic (-COOH) and a -OH groups
- To amine bonds thus releasing a carboxylic (-COOH) and -NH2 groups.
Which are the results of hydrolytic reactions?
- metabolites having much lower activity compared to the parent molecules, being more polar and subjected to further reactions.
- detoxification of several insecticides (organophosphorus compounds, carbamates, pyrethroids)
- bioactivation of some pro-drugs, such as acetylsalicylic acid to salicylic acid.
give a general overview of phase II biotransformation reactions
endogenous compounds must be activated with high-energy molecules such as UTP or ATP (general rule).
These activated endogenous groups are transferred by enzymes (collectively called transferases) to appropriate substrates. They may be transferred to:
- Compound of xenobiotic or endogenous nature (e.g., steroids, thyroid hormones, bilirubin)
- Their phase I metabolites, if the polar group is present in the backbone (-OH, -NH2, -COOH)
The conjugated metabolites are usually of larger dimensions, much more polar and less active than parent compounds.
In which cases the conjugated metabolites are more lipid, soluble and able to cross most barriers?
- Metallic mercury (e.g., coming from industrial use) may be methylated by microorganisms to methylmercury: methylmercury is much more lipid-soluble than metallic mercury and it may easily enter even the blood brain barrier.
- Morphine O-glucuronide, despite the fact it is a phase II metabolite, and it maintains all characteristics of most phase II metabolites (larger, more polar), it can cross all barriers, including the blood brain barrier thus exerting at that level pharmaco-toxicological actions.
