Toxicokinetics and biotransformation

Question 1

What is toxicokinetics?

Answer 1

The study of the quantification and determination of the time course of the disposition/ADME for a given xenobiotic.

external dose –> absorbed dose –> target dose –> effect

Question 2

Explain the one compartment model in broad terms.

Answer 2

The idea behind the one central compartment model is seeing the whole body as one homologous compartment, in which the chemical is distributed. This does not mean that the chemical is distributed evenly in all tissues, but that the changes in plasma concentration is proportional to the changes in tissue concentrations over time.

This works as a model for some substances: compounds whose toxicokinetics can be described with a one-compartment model rapidly equilibrate, or mix uniformly, between blood and the various tissues relative to the rate o elimination.

Question 3

Explain the two-compartment model in broad terms.

Answer 3

The two compartment model includes two compartments, one central and one (or more) peripheral compartment(s). The two compartment model treats the blood (central) and tissues (peripheral) as two separate units, each with its own rate of absorption/elimination.

This is used for chemicals with more than one distribution phase, meaning the distribution into the peripheral compartment is at a different rate than into the central compartment. The more compartments, the closer to the true kinetics of the compound but also the more complex the model.

Question 4

The basis of toxicokinetics is looking at plasma concentration and inferring data from there, name two things we use toxicokinetics for?

Answer 4

• With toxicokinetics, we can describe the relationship between external (e.g. air, diet) and biological exposure (e.g. plasma, tissue, urine).
• Using toxicokinetics, we can inform extrapolations from in vitro to in vivo (i.e. IVIVE), cross-species, and provide estimates for in vitro testing based on observed biomonitoring data (i.e. reverse dosimetry).

Question 5

There are two types of kinetics for a compound, which and what do they mean?

Answer 5

Zero order and first order kinetics.

• Zero order kinetics: A constant amount [mg] of chemical
  in the body is eliminated per unit of time, which gives a linear appearance when plotted. Zero order kinetics is concentration-independent as the dose doesn’t matter for the elimination rate. A physiological example of this would be alcohol metabolization, where ADH can only metabolize a certain amount of alcohol per unit of time, and it does so at a steady, maximal rate. As long as alcohol is present in the bloodstream, ADH continues to work at its maximum capacity.
• First order kinetics: A constant fraction (percentage) of a chemical is eliminated per unit time, which gives a steeper slope initially and then more shallow slope when plotted. First order kinetics is concentration-dependent, as the concentration decreases, so does the rate of elimination. A physiological example of this would be that if you have more toxicant in your system, the probability of linking with an enzyme is higher, so the higher the concentration -> the higher the rate of elimination.

Question 6

What is clearance (CL)?

Answer 6

Clearance describes the rate in which a chemical/toxicant is cleared from a certain volume over time and therefore has the unit mL/min. For example, a clearance of 100 mL/min would mean that 100 mL of blood/fluid containing toxicant would be cleared every minute. A high clearance means that the elimination is efficient (and usually fast) while a low clearance means that the elimination is inefficient (and often slow). Each organ has its own clearance rate and the overall clearance is each organ’s clearance added.

Question 7

What is a non-compartment model and what parameters can we get out of it?

Answer 7

A non compartment model is based on observed concentrations over time – commonly used in drug development. We administer a known dose (i.e. IV or infusion administration), and then plasma/serum levels are monitored for a set amount of time for a set of specific individuals. This allows us to estimate:
- Cmax maximum body concentration
- Tmax: time point that Cmax is reached
- AUC (area under the curve): total exposure of the target tissue during
time of exposure.
- CL: clearance: how much of the compound that’s cleared in a volume/time.
- half-life (i.e. the amount of time needed for a compound to decrease by half compared to Cmax/initial concentration)

Question 8

What is the difference between a one-compartment model and a non-compartment model?

Answer 8

Both a one-compartment model and a non-compartment model assumes that the body is one homologous compartment, but in one compartment models you base it on historic/pre-recorded data while in a non-compartment model is used to do estimates directly from the observed data requiring no historical knowledge of the pharmacokinetic characteristics of the drug in the body. This is cheaper and less complex.

Question 9

What is the half-life (T1/2) of a toxin?

Answer 9

The half life of a toxin is the time it takes for half of the initial toxin concentration to be eliminated (that includes any process that leads to the toxin no longer showing up in plasma) and is thus dependent on both volume of distribution and clearance.

Question 10

Remember to study arithmetical problems for toxicokinetics!!!

Answer 10

Remember to study arithmetical problems for toxicokinetics!!!

Question 11

Propose two potential advantages and two potential disadvantages on using non-compartmental models?

Answer 11

Advantages:
- Model-independent – no assumptions about body physiology (e.g. tissue volume, cardiac output) or chemical- specific parameters (e.g. partitioning coefficient, binding affinity)
- Less complex algebraic equations
- Fast and more cost-efficient

Disadvantages:
- No extrapolation between doses, time points, experimental models, etc.
- Does not reflect population characteristics with persons of different age, ethnicity, disease status, genetic polymorphism, etc.
- No information about effects, just ADME over time
- No specific information about levels in different organs/tissues
- Exposure characteristic is often missing in environmental studies

Question 12

Another type of toxicokinetic model is PBPK modelling. What is it and what is it used for?

Answer 12

PBPK = Physiologically based pharmacokinetic modelling. PBPK modelling is used for predictive purposes, and it takes both chemical and physiologic factors into account, like absorption and clearance in different organs (chemical) and blood flow, pH and protein/lipid fraction of cells (physiological), to make it as close to the truth as possible.

Question 13

Name three chemical-specific and three physiology-specific parameters that would affect ADME in a PBPK model.

Answer 13

Chemical specific:
- ADME into different organs
- compound specific factors such as lipophilicity, solubility
- enzyme affinity

Physiology specific:
- blood flow in organ
- pH
- Organ volume

Question 14

What can we use toxicokinetic modelling for?

Answer 14

1. Extrapolations
   - From cells to animals to man (IVIVE)
   - From high dose to low dose
   - Back-calculate internal exposure to intake
   - Between routes of exposure (oral, inhalation, skin)
2. A tool in biomonitoring
3. A tool for building and testing hypothesis
4. Better understanding of factors that contribute to (risk of) toxicity

Question 15

What is biotransformation?

Answer 15

Biotransformation is the metabolic conversion of endogenous and xenobiotic chemicals to more water-soluble compounds.

Question 16

What is the difference between biotransformation and metabolism?

Answer 16

Biotransformation is a subset of metabolism, with particular focus on xenobiotics as exogenous compounds, not nutrients (which are converted by metabolism. So you can say that biotransformation is metabolism but not that metabolism is biotransformation.

Question 17

What is xenobiotics?

Answer 17

Xenobiotics = Substances that are foreign to, and exert adverse physiological responses within, biological systems.

Question 18

We know that biotransformation reactions result in more water soluble metabolites, but what is important to remember in regards to toxicity of the metabolites?

Answer 18

Biotransformation can result in metabolites that are less/equal or MORE toxic than the reactants.

Question 19

Why are lipophilic xenobiotics a problem? Name four issues.

Answer 19

Lipophilic compounds are easily absorbed but poorly excreted!

1. They easily diffuse through membranes and are taken up by tissues and organs
2. They are deposited in fat depots
3. They bind to plasma proteins (results in less filtration in the kidneys)
4. They are reabsorbed in intestines and kidneys

Question 20

Biotransformation reactions happen in two phases, which phases and what characterizes the reactions in each phase?

Answer 20

Biotransformation reactions have two phases, phase 1 reactions and phase 2 reactions.

• Phase 1 reactions break down big molecules into smaller metabolites (primary metabolites)
• Phase 2 reactions bind together smaller metabolites and endogenous compounds, resulting in bigger, more water soluble metabolites (intermediate metabolites)

Primary metabolites can be excreted directly too if water soluble enough.

Question 21

Which reactions are included in the phase 1 reactions?

Answer 21

Phase 1 reactions include:
- Hydrolysis: Water molecule breaks bond
- Oxidation: Oxidating agent “steals” electrons which breaks bonds
- Reduction: Reducing agent “gives” electrons which breaks bonds

Question 22

What is the main purpose of the phase 1 reactions?

Answer 22

The purpose of the phase 1 reactions is to expose a functional group, that can later get involved in the conjugating phase 2 reactions.

Question 23

Which reactions are included in the phase 2 reactions?

Answer 23

Phase 2 reactions are conjugating reactions:
- Glucuronidation (big increase in WS)
- Sulfonation (big increase in WS)
- Acetylation (low increase in WS)
- Methylation (low increase in WS)
- Glutathione conjugation (big increase in WS)
- Conjugation with amino acids

Addition of these groups facilitates excretion! (textbok from p 111 if you want to read more)

Question 24

Which is the main organ where biotransformation happens?

Answer 24

The liver is the main organ for biotransformation.

The Lung, kidneys and intestines are somewhat active in biotransformation and the skin have a minor role.

Question 25

Which four things are important to consider when looking at biotransformation?

Answer 25

• Which compound is converted?
• What are the resulting products?
• Which enzyme participates in this conversion?
• In which organs biotransformation takes place?

Question 26

Which enzyme system is deemed the most important for xenobiotic biotransformation? Where in the cell is it located?

Answer 26

The cytochrome P450 system! (CYP)

CYPs are mainly found in the smooth endoplasmic reticulum (ER) and some in the inner membrane of mitochondria.

Question 27

Name two enzymes that catalyze an hydrolysis reaction and what kind of xenobiotics act as substrate.

Answer 27

• Carboxylesterases: amides and esters as substrate (activates pharmacological compounds and can form toxic and tumorigenic
  metabolites)
• Cholinesterases: organophosphates as substrate (limit the toxicity of organophosphates)
• Alkaline Phosphatases: Activates many prodrugs such as aspirin through hydrolysis.
• Peptidases: hydrolyses peptides such as hormones
• Epoxide Hydrolases: hydrolyze unstable and chemically reactive, mutagenic and carcinogenic epoxides to less reactive, water soluble
  compounds.

Question 28

Name one enzyme that catalyze a reduction reaction and what kind of xenobiotics act as substrate.

Answer 28

• Azo- and Nitro- reductase: Reduce azo- and nitro- groups by braking N=N bond in xenobiotics (in low O2 environment)
• Carbonyl reductase: Reduces aldehydes or ketones to primary or secondary alcohols.
• Sulfoxide reductase: Reduces sulfoxide produced by CYP450 enzymes into primary/secondary alcohols.

Question 29

Name one enzyme that catalyze an oxidation reaction and what kind of xenobiotics act as substrate.

Answer 29

• Alcohol, Aldehyde and Dihydrodiol dehydrogenases: Converts alcohols into aldehydes.
• Monoamine Oxidases: Converts monoamines, incl. Serotonin and
  xenobiotics into ammonia or primary amide + aldehyde.
• CYP450: A LOT of substrates!, caffeine for example.

Question 30

What three things can cause inhibition of P450 activity?

Answer 30

• Mutations: a genetic mutation that either blocks the synthesis of a CYP enzyme or leads to the synthesis of a catalytically compromised, inactive, or unstable enzyme, which gives rise to the poor and intermediate metabolizer genotypes.
• Virus/inflammation: exposure to an environmental factor (such as an infectious disease or an inflammatory process) that suppresses CYP enzyme expression.
• Xenobiotic exposure: exposure to a xenobiotic that inhibits or inactivates a preexisting CYP enzyme. By inhibiting cytochrome P450, one drug can impair the biotransformation of another, which may lead to an exaggerated toxicologic response to the second drug.

Environmental factors known to affect CYP levels include medications, foods, social habits (e.g., a coho consumption and cigarette smoking), and disease status (diabetes, inflammation, viral and bacterial infection, hyperthyroidism, and hypothyroidism)

Question 31

What two things can induce/lead to increased CYP enzyme activity?

Answer 31

• Gene duplication: gene duplication in genes regulating CYPs can lead to an overexpression of a CYP enzyme.
• Xenobiotic exposure: exposure to drugs and other xenobiotics that induce the synthesis of CYPs by acting as a ligand for a receptor that dimerizes with a transcription factor that induce CYP expression.
• Activation of an already existing enzyme (a little unclear?)

Question 32

What is the mechanism of xenobiotic exposure leading to induction of CYP1A?

Answer 32

The xenobiotic binds to AhR (aryl-hydrocarbon receptor) in cytosol, the complex translocates to the nucleus where it dimerizes with an ARNT (aryl hydrocarbon receptor nuclear translocator) to form a transcription factor that bind to DNA to induce gene expression of CYP1A.