How Xenobiotics Cause Toxicity

Some xenobiotics cause toxicity by disrupting normal cell functions: Bind and damage proteins (structural, enzymes) Bind and damage DNA (mutations)

Bind and damage lipids

React in the cell with oxygen to form free radicals which damage lipid, protein, and DNA

Types of Toxic Effects

Death - arsenic, cyanide Organ Damage - ozone, lead

Mutagenesis - UV light
Carcinogenesis - benzene, asbestos Teratogenesis - thalidomide

Metabolism of xenobiotics Chemical transformation of xenobiotics Occurs in mostly in liver (enzymatic processes) Convertion into more hydrophil. subst. - excretion urine

May convert procarcinogens into cytotox., mutagenic compounds

Different persons may have differences in metabolism (genetic diff., physiol. factors) Metabolism of one xenobiotic may influence metab. of another Xenobiotics Drugs Other foreign non-essential compounds Metabolism in non-hepatic tissue Intestine mucosa Kidney Lung Bacteria in GI-tract

First-pass metabolism: Xenobiotic metabolized before reaching general circulation

Introduction
Purpose
Converts lipophilic to hydrophilic compounds Facilitates excretion

Consequences
Detoxification Metabolic activation

Biotransformation
Potentially toxic xenobiotic
Relatively harmless

Detoxification

Metabolic activation

Inactive metabolite

Reactive intermediate

Converting lipophilic to water soluble compounds

Lipophilic

Xenobiotic
Phase I - Activation Reactive intermediate Phase II - Conjugation Conjugate

(non-polar)

Excretion

Water soluble (polar)

Phase I
introduction of functional group
hydrophilicity increases slightly may inactivate or activate original compound major player is CYP or mixed function oxygenase (MFO) system in conjunction with NAD(P)H location of reactions is smooth endoplasmic reticulum

Phase II
conjugation with endogenous molecules
(GSH, glycine, cystein, glucuronic acid)

hydrophilicity increases substantially neutralization of active metabolic intermediates facilitation of elimination location of reactions is cytoplasm

Comparing Phase I & Phase II

Enzyme Types of reactions Phase I Hydrolysis Oxidation Reduction Small Exposes functional group May result in metabolic activation Phase II Conjugations

Increase in hydrophilicity General mechanism Consquences

Large Polar compound added to functional group Facilitates excretion

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Phase I reactions
Oxidation
Hydroxylation (addition of -OH group) N- and O- Dealkylation (removal of -CH side chains) Deamination (removal of -NH side chains) Epoxidation (formation of epoxides) Oxygen addition (sulfoxidation, N-oxidation) Hydrogen removal

O C C

epoxide

Reduction
Hydrogen addition (unsaturated bonds to saturated) Donor molecules include GSH, FAD, NAD(P)H Oxygen removal

Hydrolysis
Splitting of amide and ester bonds

Drug Metabolism

Extrahepatic microsomal enzymes (oxidation, conjugation)

Hepatic microsomal enzymes (oxidation, conjugation)

Hepatic non-microsomal enzymes (acetylation, sulfation,GSH, alcohol/aldehyde dehydrogenase, hydrolysis, ox/red)

Metabolic enzymes
1. Microsomal:
1. 2. CYP450 monooxygenases Flavin monooxygenase

2. Non-microsomal
1. 2. 3.
1. 2. 3.

Alcohol dehydrogenase Aldehyde dehydrogenase Monoamine and diamine oxidases

Esterases and Amidases Prostaglandin synthase Peroxidases

3. Both

Liver Microsomal System

Oxidative Reactions: Cytochrome P450 mediated

Cytochrome P450 monooxygenases

A superfamily of heme containing phase I enzymes which oxidize endogenous and exogenous substrates Complex between CO and cyt P450 absorbs light maximally at 450 nm Overall reaction proceeds by catalytic cycle: RH+O2+H++NADPH ROH+H2O+NADP+ High catalytic versatility Highest concentration in ER (microsome) Present in virtually all tissues

Cytochrome P450 (CYP) Mixed Function Oxidases (MFO)

Located in many tissues but highly in liver ER Human: 16 gene families CYP 1,2,3 perform drug metabolism >48 genes sequenced Key forms: CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 Highly inducible
Alcohol Dioxin/PCBs Barbiturates CYP2E1 CYP1A CYP2B

CYP genes have multiple alleles (2D6 has 53, and 2E1 has 13)

Cytochrome P450 contains heme.

N Fe N

N N

Heme

The heme group is a cofactor which contains iron.

Different forms of heme are found in hemoglobins and proteins known as cytochromes.
The iron in a heme can bind oxygen and transfer electrons.

Cytochrome P450 Cytochrome P450s are integral membrane proteins found in the endoplasmic reticulum-membrane network in cells

NADP+

Drug CYP R-Ase ePC

CYP
Drug

Fe+3 Drug OH CYP Fe+3 Drug OH e-

NADPH CO
CYP-Fe+2 Drug

CO hu

CYP Fe+2 Drug O2 O2 CYP Fe+2 Drug

H2O 2H+

Electron flow in microsomal drug oxidizing system

CYP Nomenclature
Families - CYP plus arabic numeral (>40% homology of amino acid sequence, eg. CYP1) Subfamily - 40-55% homology of amino acid sequence; eg. CYP1A Subfamily - additional arabic numeral when more than 1 member has been identified; eg. CYP1A2 Italics indicate gene (CYP1A2); regular font for enzyme

Cytochrome P450 Isoforms (CYPs) - An Overview

NADPH + H+ + O2 + Drug NADP+ + H2O + Oxidized Drug Carbon monoxide binds to the reduced Fe(II) heme and absorbs at 450 nm (origin of enzyme family name) CYP monooxygenase enzyme family is major catalyst of drug and endogenous compound oxidations in liver, kidney, G.I. tract, skin, lungs Oxidative reactions require the CYP heme protein, the reductase, NADPH, phosphatidylcholine and molecular oxygen CYPs are in smooth endoplasmic reticulum in close association with NADPH-CYP reductase in 10/1 ratio The reductase serves as the electron source for the oxidative reaction cycle

Participation of the CYP Enzymes in Metabolism of Some Clinically Important Drugs

CYP Enzyme Examples of substrates
1A1 1A2 2A6 2B6 2C-family Caffeine, Testosterone, R-Warfarin Acetaminophen, Caffeine, Phenacetin, R-Warfarin 17-Estradiol, Testosterone Cyclophosphamide, Erythromycin, Testosterone Acetaminophen, Tolbutamide (2C9); Hexobarbital, SWarfarin (2C9,19); Phenytoin, Testosterone, R- Warfarin, Zidovudine (2C8,9,19); Acetaminophen, Caffeine, Chlorzoxazone, Halothane Acetaminophen, Codeine, Debrisoquine Acetaminophen, Caffeine, Carbamazepine, Codeine, Cortisol, Erythromycin, Cyclophosphamide, S- and RWarfarin, Phenytoin, Testosterone, Halothane, Zidovudine Adapted from: S. Rendic Drug Metab Rev 34: 83-448, 2002

2E1 2D6 3A4

CYP Biotransformations
Chemically diverse small molecules are converted, generally to more polar compounds Reactions include:
Aliphatic hydroxylation, aromatic hydroxylation Dealkylation (N-,O-, S-) N-oxidation, S-oxidation Deamination Dehalogenation

The reaction of cytochrome P450 can also produce a toxic compound:

Metabolism of acetaminophen (Tylenol)

O C HN CH3

OH Acetaminophen
At normal doses, sulfate or a sugar is attached to OH and the drug is easily removed by the kidney.

At high doses, this pathway cannot keep up and a liver P450 converts acetaminophen to a toxic metabolite which causes liver damage.

Non-CYP Drug Biotransformations

Oxidations Hydrolyses Conjugation (Phase 2 Rxs)
Major Conjugation Reactions Glucuronidation (high capacity) Sulfation (low capacity) Acetylation (variable capacity)
Examples: Procainamide, Isoniazid

Other Conjugation Reactions: O-Methylation, SMethylation, Amino Acid Conjugation (glycine, taurine, glutathione) Many conjugation enzymes exhibit polymorphism

Non-CYP drug oxidations

Monoamine Oxidase (MAO), Diamine Oxidase (DAO) - MAO (mitochondrial) oxidatively deaminates endogenous substrates including neurotransmitters (dopamine, serotonin, norepinephrine, epinephrine); drugs designed to inhibit MAO used to affect balance of CNS neurotransmitters (L-DOPA); MPTP converted to toxin MPP+ through MAO-B. DAO substrates include histamine and polyamines. Alcohol & Aldehyde Dehydrogenase - non-specific enzymes found in soluble fraction of liver; ethanol metabolism Xanthine Oxidase - converts hypoxanthine to xanthine, and then to uric acid. Drug substrates include theophylline, 6mercaptopurine. Allopurinol is substrate and inhibitor of xanthine oxidase; delays metabolism of other substrates; effective for treatment of gout.

Non-CYP drug oxidations

Flavin Monooxygenases Family of enzymes that catalyze oxygenation of nitrogen, phosphorus, sulfur particularly facile formation of N-oxides Different FMO isoforms have been isolated from liver, lung Complete structures defined

Require

molecular

oxygen,

NADPH,

flavin

adenosine

dinucleotide (FAD) FMOs are heat labile and metal-free, unlike CYPs Factors affecting FMOs (diet, drugs, sex) not as highly studied as CYPs

Several important neurotransmitters are amines.

HO HO NH3 dopamine HO OH HO NH3 norepinephrine HO NH3 HO NH2 CH3 epinephrine N H serotonin NH3

HO

These are released into the blood where they bind to receptors on the surface of cells.

To ensure that the action of amine neurotransmitters is short, they are destroyed by the enzyme monoamine oxidase.

Monoamines also come from the diet

Tyramine is formed from tyrosine. If blood levels rise they disrupt catecholamine metabolism, leading to possibly fatal hypertension. Monoamine oxidase prevents this.

HO NH3 tyramine

Foods containing high levels of tyramine: smoked, aged, or pickled meat or fish; sauerkraut aged cheeses (e.g., swiss, cheddar, blue, boursault, camembert, emmenthaler, stilton); yeast extracts fava beans beef or chicken liver aged sausages (e.g., bologna, pepperoni, salami, summer sausage) game meats (e.g., venison, rabbit) red wines (e.g., chianti, sherry).

Monoamine oxidase

An integral membrane protein in liver mitochondria. Oxidizes amines to aldehydes:

HO HO NH3

O2

HO HO O + NH4

Monoamine oxidase contains FAD as a cofactor

R' N N

O NH

Flavin Adenosine Dinucleotide

Synthesized from the vitamin riboflavin Used in electron transfer reactions

Monoamine oxidase inhibitors

Monoamine oxidase (MAO) inhibitors are used as antidepressants and to treat eating disorders. They keep serotonin levels up. Serotonin controls mood. MAO inhibitors can cause overdoses: No longer break down tyramine from diet. High levels of tryptophan in diet can result in serotonin levels becoming too high. Will prevent metabolism of drugs that are amines. As a result MAO inhibitors can result in serious side effects when used with other drugs. MAO inhibitors have been replaced with drugs which prevent the binding of serotonin to cells, such as Prozac. These do not affect the metabolism of other amines, so that there are far fewer side effects.

Phase I: Hydrolysis
Carboxyesterases & peptidases
hydrolysis of esters eg: valacyclovir, midodrine hydrolysis of peptide bonds e.g.: insulin (peptide)

Epoxide hydrolase
H2O added to epoxides eg: carbamazepine
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Phase I: Reductions
Azo reduction
N=N to 2 -NH2 groups eg: prontosil to sulfanilamide

Nitro reduction
N=O to one -NH2 group eg: 2,6-dinitrotoluene activation

Reductions
Carbonyl reduction
Alcohol dehydrogenase (ADH)
Chloral hydrate is reduced to trichlorothanol

Disulfide reduction
First step in disulfiram metabolism

Sulfoxide reduction
NSAID prodrug Sulindac converted to active sulfide moiety

Reductions
Quinone reduction
Cytosolic flavoprotein NAD(P)H quinone oxidoreductase
two-electron reduction, no oxidative stress high in tumor cells; activates diaziquone to more potent form

Flavoprotein P450-reductase
one-electron reduction, produces superoxide ions metabolic activation of paraquat, doxorubicin

Reductions
Dehalogenation
Reductive (H replaces X)
Enhances CCl4 toxicity by forming free radicals

Oxidative (X and H replaced with =O)

Causes halothane hepatitis via reactive acylhalide intermediates

Dehydrodechlorination (2 Xs removed, form C=C)

DDT to DDE

Phase I: Oxidation-Reduction
Alcohol dehydrogenase
Alcohols to aldehydes Genetic polymorphism; Asians metabolize alcohol rapidly Inhibited by ranitidine, cimetidine, aspirin

Aldehyde dehydrogenase
Aldehydes to carboxylic acids Inhibited by disulfiram

Phase I: Monooxygenases
Monoamine oxidase
Primaquine, haloperidol, tryptophan are substrates Activates 1-methyl-4-phenyl-1,2,5,6tetrahydropyridine (MPTP) to neurotoxic toxic metabolite in nerve tissue, resulting in Parkinsonian-like symptoms

Monooxygenases
Peroxidases couple oxidation to reduction of H2O2 & lipid hydroperoxidase
Prostaglandin H synthetase (prostaglandin metabolism)
Causes nephrotoxicity by activating aflatoxin B1, acetaminophen to DNA-binding compounds

Lactoperoxidase (mammary gland) Myleoperoxidase (bone marrow)

Causes bone marrow disorders by activating benzene to DNA-reactive compound

Monooxygenases
Flavin-containing mono-oxygenases
Generally results in detoxification Microsomal enzymes Substrates: nicotine, cimetidine, chlopromazine, imipramine Repressed rather than induced by phenobarbital, 3-methylcholanthrene

Phase II reactions
Glycoside conjugation - glucuronidation Sulfate - sulfation Glutathione (GSH) Methylation Acylation
Acetylation Amino acid conjugation Deacetylation

Phosphate conjugation

Phase II: Glucuronidation

Major Phase II pathway in mammals UDP-glucuronyltransferase forms O-, N-, S-, C- glucuronides; six forms in human liver
Cofactor is UDP-glucuronic acid Inducers: phenobarbital, indoles, 3methylcholanthrene, cigarette smoking Substrates include dextrophan, methadone, morphine, p-nitrophenol, valproic acid, NSAIDS, bilirubin, steroid hormones
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Glucuronidation & betaglucuronidase

Conjugates excreted in bile or urine beta-glucuronidase from gut microflora cleaves glucuronic acid Aglycone can be reabsorbed & undergo enterohepatic recycling

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Phase 2: Conjugation

Glucuronic acid conjugation

Substrates: Alchohols Phenols Amines Sulfides

RXH: Xenobiotic / Phase 1 metabolite

X R HO OH OH O OH OH CO2H O OH OH O OH P H N O P O N O CO2H X O OH OH OH R

Glucose

UDP-Glucuronate HO OH

Carboxylic acids
1,3-Dicarbonyls
RXH CO2H X O OH OH OH R Kidney Urive excretion CO2H X O OH OH OH R

Bile (galle) Reabsorb R Intestine

Poor reabsorb.

Entro-hepatic recycling Important for many hormones etc

RXH

CO2H X O OH OH OH

Glucuronidation & genetic polymorphism

Crigler-Nijar syndrome (severe): inactive enzyme; severe hyperbilirubinemia; inducers have no effect Gilberts syndrome (mild): reduced enzyme activity; mild hyperbilirubinemia; phenobarbital increases rate of bilirubin glucuronidation to normal Patients can glucuronidate p-nitrophenol, morphine, chloroamphenicol
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Phase II: Sulfation

Sulfotransferases are widely-distributed enzymes Cofactor is 3-phosphoadenosine-5phosphosulfate (PAPS) Produce highly water-soluble sulfate esters, eliminated in urine, bile Xenobiotics & endogenous compounds are sulfated (phenols, catechols, amines, hydroxylamines)
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Sulfation
Sulfation is a high affinity, low capacity pathway
Glucuronidation is low affinity, high capacity

Capacity limited by low PAPS levels

Acetaminophen undergoes both sulfation and glucuronidation At low doses sulfation predominates At high doses, glucuronidation predominates
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Phase II: Acetylation

Major route of biotransformation for aromatic amines, hydrazines Generally decreases water solubility N-acetyltransferase (NAT)
Cofactor is AcetylCoenzyme A

Humans express two forms Substrates include sulfanilamide, isoniazid, dapsone

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Phase II:Amino Acid Conjugation

Alternative to glucuronidation Two principle pathways
-COOH group of substrate conjugated with NH2 of glycine, serine, glutamine, requiring CoA activation
e.g: conjugation of benzoic acid with glycine to form hippuric acid

Aromatic -NH2 or NHOH conjugated with COOH of serine, proline, requiring ATP activation
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and decomposes hydrogen peroxide