Principles of Toxicology Exam 2

Biotransformation (Metabolism in ADME)

enzymatic process of chemical modification that generally changes the physiochemical properties of a xenobiotic from those that favor absorption and distribution, to those that favor elimination (i.e., lipophilic → hydrophilic)

What is the exception to our definition of biotransformation?

elimination of volatile compounds by exhalation

Parent Xenobiotic

form that is absorbed and may be a substrate for biotransformation

Ultimate Xenobiotic

form that causes a change in the body, may be a product of biotransformation

Can the ultimate xenobiotic also be the parent xenobiotic?

Yes it can

What does TPSA measure?

the number of oxygen and nitrogen atoms that bind water by functioning as hydrogen
donors or acceptors

What is the relationship between passive diffusion and TPSA? (increasing/decreasing)

Rate of passive diffusion decreases with increasing TPSA

What value of TPSA restricts passive diffusion? (acids)

> 75 Angstrom

What value of TPSA restricts passive diffusion? (bases)

> 100 Angstrom

What is the process of xenobiotic biotransformation?

the process of converting lipophilic chemicals which are
readily absorbed from the GI and other sites into hydrophilic chemicals, which are readily excreted in
urine or bile

Hydrolysis Reactions

break covalent bonds in substrates

Reduction Reactions

transfer electron(s) to substrates

Oxidation Reactions

Remove electron(s) from substrate

Conjugation Reactions

Covalent attachment of chemical groups to substrate. Examples include: sulfonation, acetylation , methylation, conjugation with AA, etc.

Hydrolysis, reduction and oxidation expose or introduce a _____________?

functional group that can be converted into a water-soluble cojugate

(T/F): Phase I reactions always happen first

False; not always

What reaction(s) cause a modest increase in water solubility?

Oxidation, reduction, hydrolysis, and acetylation

What reaction(s) cause a modest decrease in water solubility?

methylation

What reaction(s) cause a marked increase in water solubility?

glucuronidation, sulfonation, glutathionylation and AA conjugation

What Log P and TPSA values are required for a drug to be absorbed by passive diffusion in the GI lumen?

Log P: 0-5
TPSA: <100 Angstrom

What range of Log P values are generally insoluble in the GI lumen?

Log P >5

What range of Log P values are absorbed orally in substrate for uptake transporter?

Log P < 0

What is activation?

conversion of a benign parent xenobiotic to a biologically active (ultimate) form, or increase in toxicity of parent xenobiotic

What is inactivation?

conversion of a biologically active xenobiotic to a benign or less toxic form (detoxification)

What organ expresses the largest number and (generally) the highest concentrations of xenobiotic- metabolizing enzymes?

The liver

What do efflux transporters do in the liver?

remove xenobiotics from blood and discharge xenobiotics into bile, or discharge metabolites into blood for excretion in urine

How do efflux transporters affect cells in the small intestine?

cells express high levels of certain enzymes

UM

ulta-rapid metabolizer

EM

extensive metabolizer (normal)

IM

intermediate metabolizer

PM

poor metabolizer

T/F: individual xenobiotic-transforming enzymes are located in a single organelle

True

What two factors influence biotransformation rates?

disease state & cofactor levels

T/F: Xenobiotic transformation is accomplished by a limited number of enzymes with broad substrate specificities

True

How are enzymes/substrates named in toxicology?

The broad spectrum of substrates prevents enzymes from being named after the reaction they catalyze, but rather
in families and subfamilies, then named according to primary AA sequence

What is distribution?

the systemic movement of xenobiotics within the body from the site of absorption to the target tissue (requires circulatory system)

What factors affect the rate of distribution? (4)

• Blood flow

• Sequestration in blood or off-target tissue

• Transport from organ capillary beds into organ interstitial fluid

• Affinity for target tissue

What is Vd?

Volume of distribution, apparent volume in which the amount of chemical disperses in order to produce the observed blood concentration

What is the equation for Vd?

Vd = (amount of drug in the body)/(blood concentration of drug)

What do low and high Vd values suggest?

Low Vd: chemical distributes only to the plasma (no tissue distribution)
High Vd: chemical distributes to the total body of water

T/F: storage/sequestration can not hinder distribution

False, xenobiotics may have an affinity for tissue other than their target organ of toxicity

What are the four (4) major sites of xenobiotic sequestration?

• blood plasma

• body fat

• keratinized tissue/hair

• bone

What is a “storage depot”?

a compartment where chemical is concentrated, but not major site of toxicity

T/F: binding of a xenobiotic to its receptor is reversible (in blood plasma)

True

What is the ratio of affinity?

k_off / k_on

What is k_d?

equilibrium dissociation constant for a given xenobiotic ligand (L) and its receptor (R), a quantitative measure of binding affinity, xenobiotic concentration where 50% of binding sites are bound at equilibrium

Give the equation for k_d

Kd = [L][R]/[LR]

What do low and high values of k_d mean for affinity?

low kd = high affinity

high kd = low affinity

What are adipocytes?

Adipocytes are fat storing cells in adipose tissue

Which is more likely to be sequestered in body fat: highly or barely lipophilic xenobiotics?

Highly lipophilic xenobiotics (LogP > 1) are more likely to be sequestered (bioaccumulate) in body fat

Which two (2) organs have a high capacity for acting as storage depots?

liver and kidneys

What are osteoclasts?

bone breakdown via osteolysis, can release xenobiotics

What are osteoblasts?

bone formation via mineralization

T/F: the blood-brain-barrier is very effective against xenobiotic penetration

True

How does lipophilicity and ionization/polarization affect distribution of xenobiotics into the brain?

distribution increases as lipophilicity increases; distribution decreases as ionization decreases

What are the 3 primary routes of elimination in the body?

kidneys (urine), feces, lungs (exhalation)

What are nephrons and where are they located?

Nephrons are the functional units of the kidneys where urine formation occurs

What is the glomerulus in the nephrons?

Glomerulus: specialized capillary structure that serves as a size/charge filter due to its fenestrated (highly porous) endothelium

What does the tubule system do in nephrons?

Tubule system: modifies and concentrates the urine

What is a hydrolysis reaction?

hydrolysis: cleavage of covalent bonds by reaction with water

What are the 4 catalysts for hydrolysis reactions?

carboxylesterases, cholinesterases, paraoxoneses, and albumin

Does CES1 prefer to hydrolyze xenobiotics with small or large alcohol leaving groups?

small

Does CES2 prefer to hydrolyze xenobiotics with small or large alcohol leaving groups?

large

What is the difference between AChe and BChe?

AChe is highly selective to acetylcholine and has little to no role in metabolism while BChe hydrolyzes numerous xenobiotics like aspirin, heroine, cocaine, succinylcholine, etc.

_________ catalyzes the hydrolysis of many organophosphates, organophosphinites, aromatic carboxylic acid esters, cyclic carbonates, lactones and oxidized phosphates

paraoxonases (lactonases)

What are the 2 key features of paraoxonases?

1. contain R-SH group

2. calcium dependent

What are carboxylesterases and what organs/tissues are they found in (3)?

predominately microsomal enzymes present in liver, intestine, kidneys, other tissues

What 3 substances do carboxylesterases commonly hydrolyze?

aspirin, cocaine, heroin

What is a prodrug and what type of reaction activates it?

an inactive form of a medication that requires metabolic conversion within the body to become pharmacologically active; hydrolysis via carboxylesterases or cholinesterases

In an oxidation reaction, would an aldehyde (CHO) become an alcohol (OH) or carboxylic acid (COOH)?

carboxylic acid (COOH)

In a reduction reaction, would an aldehyde (CHO) become an alcohol (OH) or carboxylic acid (COOH)?

alcohol (OH)

What are cofactors?

molecules or ions that associate with some enzymes and are used to perform reactions

What are prosthetic groups?

a type of cofactor that are tightly bound by enzymes and are essential for enzyme function

Are NAD+/NADH and NADP+/NADPH cofactors or prosthetic groups?

cofactors

Are FAD/FADH2 and heme group/iron cofactors or prosthetic groups?

prosthetic groups

Label the missing parts of this image

1. ADH

2. ALDH

3. NAD+

4. NADH

5. NAD+

6. NADH

Which is found in the cytoplasm: ADH or ALDH?

ADH

Which is found in the mitochondria: ADH or ALDH?

ALDH

What is monooxygenation?

one atom from O 2 is incorporated into a substrate and the other is reduced to water

What organelle in which organ is the highest level of CYP observed?

liver smooth ER (present in virtually all tissues though)

What are the 5 requirements of the CYP monooxygenation reaction?

• O2

• Heme (Fe 2+/3+)

• NADPH

• NADPH - cytochrome P450 reductase

• cytochrome b5

List all the primary Phase II Conjugation Reactions (6)

1. Methylation

2. N-Acetylation

3. Glucuronidation

4. Sulfonation

5. Glutathione

6. Amino acids

What is/are the required enzyme(s) for N-Acetylation reactions?

2 N-acetyltransferases (NATs); NAT-1 and NAT-2

What cofactor is required for the enzymes in N-Acetylation?

acetyl-coenzyme A (acetyl-CoA)

What are the two functional groups that act as substrates in N-Acetylation reactions?

amine (R-NH2) and hydrazine (R-NH-NH2)

What is the typical structure of R in a N-Acetylation reaction substrate?

R is usually an aromatic ring structure

Acetyl-CoA _______ the _______ group to the N-acetylation reaction

donates; acetyl

Describe the 2 steps of a N-Acetylation reaction

Step 1: Acetyl group is covalently attached to the enzyme
Step 2: Acetyl group is transferred to the xenobiotic amine/hydrazine functional groups

What type of Phase II reaction is this?

N-Acetylation

What type of Phase II reaction is this?

N-Acetylation

What type of Phase II reaction is this?

N-Acetylation

What type of Phase II reaction is this?

N-Acetylation

What is/are the required enzyme(s) for methylation reactions?

O-, N-, S- methyltransferases

What is the cofactor for methylation?

S-adenosylmethionine (SAM)

S-adenosylmethionine (SAM) _______ a _______ group to the xenobiotic substrate

donates; methyl

What are COMT and POMT?

COMT = catechol O-methyltransferase with 2 OH groups on a benzene ring

POMT = phenol O-methyltransferase with 1 OH group on a benzene ring

T/F: methylation represents an important pathway of detoxication

True

Does a methyl group increase or decrease lipophilicity of xenobiotic substrates?

increase lipophilicity

COMT or POMT?

COMT