Lesson 4

Question 1

Which are the phase II biotransformation reactions? (list)

Answer 1

Glucoronidation, sulphation, GSH conjugation, N-acetylation, methylation

Question 2

What is a glucoronidation?

Answer 2

We add an UTP to D-glucose-1-phospate (the endogenous substrates), this result in the formation of UDPGA (uridine-diphospho-glucoronic acid).
UDPGA is conjugated with NH2/SH/OH/COOH obtaining a glucoronide

Question 3

Where can we find UGT? What about UDPGA?

Answer 3

UDPGA is present in cells, especially in liver cells, at a quite high concentration: this means that, also in the presence of low concentration of substrate, this is not a rate limiting step.

UGT is in close proximity to cytochrome P450 (it’s not cytosolic)

Question 4

which are the functional groups that undergo glucoronidation?

Answer 4

• The ammine group (NH2): produce N-glucoronides
• The hydroxyl group (OH): produce O-glucoronides
• The carboxylic group (COOH)
• SH: produce S-glucoronides

Question 5

Describe sulphation

Answer 5

It’s a far less important reaction:
- The radicals are the same but without the carboxylic acid (only OH, NH2, SH)
- The active conjugating agent is PAPS (suphate + ATP). This is less present then UDPGA
Sulphotransferases (SULT) are cytosolic enzymes present in the cytosolic fraction

Question 6

What is GSH?

Answer 6

GSH is a tripeptide extremely active against reactive radicals. It doesn’t require activation by a high energy molecule (ATP, UDP…) because is able to conjugate appropriate substrates spontaneously, without the catalysis of GSH-transferases (although at a very low rate).
GSH is rapidly synthesized and it’s normally present at a very high concentration in liver cells (higher than UGDPA).

Question 7

What is the role of GSH conjugation?

Answer 7

they’re crucial in the anti-oxidant defense of cells.
Phase I reactions are sometimes able to produce metabolites such as epoxides with high reactive activity. Particularly, we have mentioned aflatoxin B1 epoxide. GSH are scavengers of reactive species, even or oxygen radicals

Question 8

Give an example of GSH conjugation

Answer 8

CYP1A mediated the formation of aflatoxin B1 epoxide (which is a mycotoxin). This epoxide may be totally inactivated by GSTμ with the formation of GS-conjugate of aflatoxin B1. After the reaction the epoxide group, which is directly linked with the reactivity of the molecule, is no longer present.
GS-conjugates have different fates:
- In an extreme minority of cases, GS-conjugates are excreted, without any further biotransformation, via the biliary route.
- In most case, GS-conjugates are eliminated via the urinary route. If you administer a compound to an animal and then you find mercapturic acid derivates in urines, it is a proof of the occurred GS-conjugation.

Question 9

Describe the N-acetylation

Answer 9

The endogenous conjugating agent is acetyl-CoA.
We have 2 different families of N-acetyltransferases in mammalian species: NAT1 e NAT2, both are cytosolic.
The conjugable substrates are very limited, the most important substrates are arylamines drugs, especially sulfonamides.
N-acetylation can take place on 2 different -NH2 groups:
- Amine group in position 1
- Amine group in position 4
At the and we will have 2 different derivatives.

Question 10

Which phase II reaction occur to catecholamines and histamine?

Answer 10

In most cases methylation takes place on endogenous compounds, there are just few exceptions in which methylation is involved in bio transformation of exogenous compounds. The endogenous substrates are catecholamines and histamine.

Question 11

What is the pre-systemic metabolism?

Answer 11

The sum of all biotransformation reactions occurring before the compounds and the metabolites can reach the systemic circulation. It is also called first pass effect. The dosing of a drug should take into consideration it

Question 12

Define ‘bioavailability’

Answer 12

It’s the amount of the drug reaching the systemic circulation after undergoing the biotransformations

Question 13

Which is the aim of the post-systemic metabolism?

Answer 13

to save and reuse precious compounds

Question 14

How does phase I biomodifications affect the post-systemic metabolism?

Answer 14

If the compound is a metabolite with a further OH group or NH2 group added by phase I reaction, then the extent of reabsorption will be lower .

Question 15

Which are the factors affecting biotrasformations?

Answer 15

There are internal factors:
- Different according to the species
- At a sub-population level (genetic polymorphism)
- Diet
- Pathological condition
- Physio-pathological conditions (pregnancy)
- Gender
- Age
Or external factors, such as the exposure to xenobiotics or drugs (they may cause a over functioning of the DME system)

Question 16

OPEN QUESTION: describe the specie-related differences in Phase I bioreaction

Answer 16

Phase I species-related difference are quantitative.
Each specie expresses the same enzymes but the quantitative expression of the different enzymes varies.
EXAMPLES:
- CYP1A is well expressed in the liver of horses and cattle, rather than in other species. It is involved in the metabolism and biotransformation of large planar molecules, this implies that horses and cattle are much more efficient in transforming this kind of compounds, but in case of bioactivation these species are more at risk.
- AhR is involved in the CYP induction. CYP1A1 and CYP1A2 are much more expressed in cows than in sheep. Differences in the expression of CYP1A1 and CYP1A2 are directly involved in the biotransformation of dioxins and dioxin-like polychlorinated biphenyl.
- Zearalenone is an estrogenic mycotoxin, with a very weak affinity for estrogen receptors.
In the reductive biotransformation are produced much more active metabolites.
The same reaction is taking place in chicken and pigs.
Pigs produce α-zearenol (reduced metabolite) with a much more estrogenic activity respect to the counterpart (β- ZEL), both are more lipophilic than ZEA.