Lash - Toxicology IV

Question 1

Bioactivation:

Answer 1

Metabolic reaction of a xenobiotic in which the product is more toxic than the substrate

Most toxic chemicals need to undergo metabolic transformations to elicit toxicity

Question 2

Active Oxygen:

Examples:

Answer 2

Short-lived, highly reactive intermediates in the reduction of oxygen

Superoxide anion (O2∙-), hydroxyl radical (OH∙), singlet oxygen (1O2), and hydrogen peroxide

Question 3

Alkylating Agent:

Mechanism:

Answer 3

Chemicals that add alkyl groups to DNA (reaction results in misparing of bases or breaks in cs)

Formation of reactive carbonium ion (ie. CH3+) that combines with electron-rich bases in DNA
o Also frequently carcinogens or mutagens

Question 4

Covalent Binding:

Answer 4

Describes binding of toxicants or their reactive metabolites to endogenous molecules (DNA, protein or lipid) to produce stable adducts
- Involved in many forms of chronic toxicity

Question 5

Detoxification:

Answer 5

Metabolic reaction(s) that reduces the potential for adverse effects of a xenobiotic

• Normally involve an increase in water solubility (facilitates excretion and/or conjugation)
• Reduces the possibility of interactions with cellular macromolecules

Question 6

Free Radicals:

Answer 6

Molecules with unpaired electrons

• Can be produced metabolically from xenobiotics
• Extremely reactive and can react with cellular macromolecules, producing adverse effects

Question 7

Glutathione:

Answer 7

Tripeptide (γ-glutamyl-L-cysteinylglycine)
- Two primary structural features:
o Nucleophilic SH group
o γ-glutamyl peptide bond (makes molecule resistant to proteases)
- Functions in many detoxification reactions in cells

Question 8

Reactive Intermediates:

Examples:

Answer 8

Reactive Intermediates: chemical compounds produced during the metabolism of xenobiotics that are more chemically reactive than their parent compound
- Have a greater potential for adverse effects than parent compound

Examples: epoxides, quinones, free radicals, ROS, small number of conjugation products

Question 9

Treatment for APAP overdose

Answer 9

N-acetylcysteine to replete hepatic GSH levels

Question 10

Five chemical mechanisms of bioactivation

Answer 10

1. Mechanism 1: Bioactivation of xenobiotics to stable, but toxic, metabolites.
2. Mechanism 2: Biotransformation of xenobiotics to reactive, electrophilic metabolites.
3. Mechanism 3: Biotransformation of xenobiotics to free radicals.
4. Mechanism 4: Formation of reduced oxygen metabolites.
5. Mechanism 5: Metabolic derangements associated with xenobiotic transformation.

Question 11

Mechanism 1: Bioactivation of xenobiotics to stable, but toxic, metabolites
examples:

Answer 11

Bioactivation of Dicholormethane to CO:
o CH2Cl2 metabolized by cytochrome P450 enzyme to an intermediate that rearranges to give CO
o CH2CL2 is often detoxified by a GSH dependent mechanism (prevent formation of CO)

Bioactivation of Acetonitrile to Cyanide:
o Acentonitrile metabolized by cytochrome P450 to an intermediate that gives HCN
o HCN normally converted to SCN- (thiocyaniate- less toxic) using rhodanese enzyme

Question 12

Mechanism 2: Biotransformation of xenobiotics to reactive, electrophilic metabolites.

Basics:
Selectivity of interaction:

Answer 12

Basics: very common bioactivation method
- Reactive electrophiles interact with cellular nucleophiles according to Pearson’s principle of hard and soft acids and bases

Selectivity of Interaction:

• Hard electrophiles (acids) interact preferentially with hard nucleophiles (bases)
• Soft electophiles (acids) interact preferentially with soft nucleophiles (bases)

Question 13

Hard base (nucleophile) is a donor atom/molecule that has the following properties: (3 and examples)

Answer 13

a. High electronegativity
b. Low polarizability
c. Difficult to oxidize
d. Examples: Amino groups, oxygen-containing functional groups in DNA and
protein

Question 14

Soft base (nucleophile) is a donor atom/molecule that has the following properties: (3 and examples)

Answer 14

a. Low electronegativity
b. High polarizability
c. Easy to oxidize
d. Examples: Thiol group of GSH and cysteine, protein sulfhydryl groups

Question 15

Hard acid (electrophile) is an acceptor atom/molecule that has the following properties: (3 and examples)

Possible Antidotes: important to note that soft bases like GSH and N-acetylcysteine will NOT be effective because they will not react with these species

Answer 15

a. High positive charge
b. Small size
c. Lacks unshared electrons in valence shell
d. Example: Alkyl carbonium ion

Question 16

Soft acid (electrophile) is an acceptor atom/molecule that has the following properties: (3 and examples)

Possible antidotes: important to note that a soft base such as N-acetlycysteine or GSH will be effective as an antidote to these species

Answer 16

a. Low positive charge
b. Relatively large size
c. Contains unshared electron pairs in valence shell
d. Example: Michael acceptors [i.e., α,β-unsaturated carbonyl compounds with
general structure R-CH=CH-C(0)-R’]

Question 17

Acetaminophen Metabolism:

Majority undergoes:
Small amount:
Normal vs. Overdose

Answer 17

Majority undergoes glucouronidation or sulfonuration –> readily excreted

Small amount metabolized by cytochrome P450 to a quinine imine (soft electrophile)

Normal: quinone imine normally conjugated to GSH (soft nucleophile) into the body, which is then readily excreted as mercaputurate

Overdose/Low GSH: not enough GSH to conjugate to, and quinone imine is able to bind/inactivate proteins

Question 18

Pearson’s law

Answer 18

Hard electrophiles react with hard nucleophiles and soft electrophiles react with soft nucleophiles.

Question 19

Electrophiles vs. Nucleophiles

Answer 19

Electrophiles (= acids) are formed on drugs during the course of their metabolism and exhibit a hardness or softness.

Nucleophiles (= bases) are the functional groups on cellular molecules like DNA, RNA, proteins, and lipids. They too exhibit some degree of hardness or softness.

Question 20

Epoxides or thiolates interact with both:

Answer 20

epoxides or thiolates (S–) are borderline and can interact (e.g., form covalent adducts) with both hard nucleophiles (e.g., amino nitrogen on DNA) and soft nucleophiles (e.g., protein-SH group).

Question 21

Carcinogenicity and toxicity with electrophiles and nucleophiles

Answer 21

Further, if an electrophile can interact with hard nucleophilic groups on DNA, it is likely to be mutagenic and carcinogenic whereas a soft electrophile typically causes acute, high-dose toxicity by inhibition proteins but cannot cause mutations or cancer.

Question 22

Bromobenzene Metabolism:
Bioactivated by:
What can epoxides interact with?

Answer 22

Bioactivated by cytochrome P450 to give a variety of different epoxides

• Some detoxified by epoxide hydrolase enzyme
• Some are toxic

Epoxides are borderline hard/soft electrophiles

• Can interact with DNA (hard nucleophiles) –> mutagenesis
• Can interact with protein sulfhydryl groups (soft nucleophiles) –> inactivation

Question 23

Benzo(a)pyrene Metabolism:

Bioactivated by:

Answer 23

Benzo(a)pyrene Metabolism:

Bioactivated by cytochrome P450 (CYP1A1/AHH) to give a variety of epoxides

Has epoxides like bromobenzene

Question 24

Acetylaminofluorene Metabolism:
Activated by:
N-hydroxylated products

Answer 24

Acetylaminofluorene Metabolism:
Activated by P450 by either C-hydoxylation (non-toxic) or N-hydroxylation (possibly toxic)

N-hydroxylated product:

• Can be excreted (glucuronidation)
• Can undergo sulfate/acyl conjugation, and instead of forming metabolite that can be excreted, forms nitrene (very hard electrophile)
• Nitrene therefore covalently binds to DNA and acts as a mutagen/carcinogen (particularly in the liver)

Question 25

Nitrosamine Metabolism:

Bioactivated by:

Answer 25

Nitrosamine Metabolism:
Bioactivated by P450 to form intermediates that undergo spontaneous cleavage to form both
- Nitrene
- Methyl carbonium ion

(Both are hard electrophiles and can therefore bind covalently to DNA and cause mutagenesis)

Question 26

Metabolism of Nephrotoxic GSH Conjugates (Trichloroethylene):

Undergoes: (2)

Hard or soft electrophiles?

Answer 26

Undergoes glutathione conjugation in the kidneys to form a cysteine conjugate

• Can undergo classic pathway (using NAT–>excreted as mercapturate)
• Can undergo another pathway using beta-lyase enzyme to form sulfur electrophiles

Sulfur electrophiles are borderline soft/hard, and therefore cause both acute toxicity (bind proteins) as well as show some evidence for DNA binding

Question 27

MECHANISM 3- FREE RADICALS:

Basics:

Answer 27

MECHANISM 3- FREE RADICALS:

Basics: can have C, N, O or S base

Question 28

Reactions of free radicals:

Answer 28

Initiation: formation of free radical
One-electron oxidation (cationic free radical)
- R –> R+∙ + e
One-electron reduction (anionic free radical)
- R + e –> R-∙
Homolytic sigma-bond cleavage (2 neutral radicals)
- R-R –> R∙ + R∙

Propagation: where potential damage occurs
Abstraction of H atoms (ie. from lipids/proteins)
-R∙ + R1H –> RH + R1∙
Addition (ie. to lipid/protein)
- R∙ + R1 –> R1-R∙

Termination:
Dimerization back to neutral species (to original non-radical species)
Disproportionation (to 2 new non-radical species)
- R1∙ + R1∙ –> R2 + R3
Reaction with antioxidant by H abstraction (ie. vitamin E; antioxidant radical formed is not unstable)

Question 29

Reactions of free radicals:

Example

Answer 29

CCl4 Metabolism:

Bioactivated by P450 (CYP2E1) to free radicals that can bind to lipid and other species

Question 30

Mechanism 4: Formation of reduced oxygen metabolites.

Sequential Reduction of Oxygen:

Answer 30

O2 –> HO2∙ (perhydroxyl radical), which can go 2 ways

o Formation of H+ + O2-∙ (superoxide anion)
o Formation of H2O2 –> HO∙ (hydroxyl radical) –> H2O

Question 31

ROS of Interest: (3)

Answer 31

1. Superoxide Anion (O2-∙): formed in many autoxidation reactions (ie. flavoproteins, redox cycling)
2. Hydrogen Peroxide: 2 electron reduction state formed from superoxide anion by dismutation or directly from O2
3. Hydroxyl Radical (HO∙): 3 electron reduction state formed by Fenton reaction or Haber-Weiss reaction
   o HIGHLY reactive: most potent of all ROS

Question 32

Intracellular Sources of ROS:

Answer 32

Nonenzymatic (Autoxidation):

• Ubisemiquinone oxidation
• Reactions with Fe2+ prosthetic groups of cytochromes, Hb, Mb

Enzymatic:

• H2O2 production (ie. flavin or copper-containing oxidases)
• Superoxide anion production (ie. flavoprotein dehydrogenases, copper-containing oxidases)

Question 33

• Examples of Redox Cycling:

Answer 33

Paraquat:

Menadione (Quinone):

Nitro Anion Free Radicals:

Question 34

Paraquat:

Answer 34

o Flavoprotein reduces to a radical that spontaneously reacts with O2 to regenerate oxidized form
o Produces superoxide anion in the process
o Occurs in the lungs of people exposed to excessive amounts lead to lung fibrosis

Question 35

Menadione (Quinone):

reducing agents:

Protective mechanism exists due to action of enzyme:

Answer 35

o Quinone –> Semiquinone (reduced radical) –> Hydroquinone (2 reduction radical)

• All occur using flavoproteins as reducing agents
• Both semiquinone and hydroquinone can spontaneously react with O2 to regenerate oxidized form (SQ –> Q; HQ –> SQ)
• Produces superoxide anion in the process

Protective mechanism exists due to action of enzyme DT-Diaphorase

• Double reduces quinone all the way to the hydroquinone, which is less reaction with oxygen than the semiquinone
• This results in less superoxide anion being produced

Question 36

Nitro Anion Free Radicals:

Produce:

Can cause production of:

Answer 36

Nitro Anion Free Radicals:
o Nitrogen compounds can undergo redox cycling to produce nitro anion free radicals

Toxic on their own

Can also cause production of nitroso products that can be even more toxic than radical itself

Question 37

MECHANISM 5- METABOLIC DERANGEMENTS ASSOCIATED WITH XENOBIOTIC TRANSFORMATION:
• Basics:

Answer 37

Basics: metabolism of these compounds results in the production of a inhibitor of a metabolic pathway or in the depletion of metabolic intermediate/coenzyme

Question 38

MECHANISM 5- METABOLIC DERANGEMENTS ASSOCIATED WITH XENOBIOTIC TRANSFORMATION:
Examples:

Answer 38

Galactosamine
Ethionine:
Fructose
Fluoroacetate (Lethal Synthesis)

Question 39

Galactosamine:

hepatotoxic due to:

Reversal:

Answer 39

Hepatotoxic due to increased synthesis of UDP-amino sugars and depletion of UTP

Reversal: addition of uridine removes toxic effects of galactosamine

Question 40

Ethionine:

Damages:

Functions affected:

Reversal:

Answer 40

Produces liver damage in a number of ways by acting as a specific antagonist of methionine through formation of S-adenosylethionine (cannot participate in methylation reactions)

Functions affected: inhibits RNA and protein synthesis, decreases glycogen synthesis, decreases phosphorylase activity, decrease cellular ATP and GSH

Reversal: giving methionine or ATP

Question 41

Fructose:

Hepatotoxic via:

Answer 41

Hepatotoxic via depletion of cellular ATP concentrations due to rapid metabolism to fructose-1-P

Question 42

Fluoroacetate (Lethal Synthesis):

simulates acetate –>? which combines with ? to form ?

Fluorocitrate inhibits ?, which is the next enzyme in the TCA cycle

Leads to:

Answer 42

Fluoroacetate (Lethal Synthesis): simulates acetate –> fluoracetyl CoA, which combines with oxaloacetate to form fluorocitrate

Fluorocitrate inhibits aconitase, which is the next enzyme in the TCA cycle

Leads to large accumulation of citrate and disruption of mitochondrial energy supply

Question 43

Lethal Synthesis

Answer 43

Describes the toxicity of fluoroacetate. In general, the term is used to describe the process by which a toxicant, similar in structure to an endogenous substrate, is incorporated into the same metabolic pathway as the endogenous substrate, ultimately being transformed into a toxic or lethal product.