Induction

• Increased transcription• Increased protein synthesis• Enhanced stability of protein• Synthesis of enzyme with higher catalytic

activityInducible forms of CYP: CYP1A1 (PAH), CYP2B, CYP3A4 (PB), CYP2E1 (EtOH)

Constitutive: CYP2Ahttp://medicine.iupui.edu/flockhart/table.htm

Example:Ah-locus mediated induction

AhR, receptor in cytoplasm, binds ligand: eg PAHs, TCDD, some PCBs

Bound AhR loses 2 heat-shock proteins (hsp90), becomes phosphorylated

Activated bound AhR migrates to nucleus, forms complex with Ah receptor nuclear translocation factor Arnt

AhR-Arnt complex binds to regulatory sequences in DNA (DRE, dioxin-responsive elements)

Transcription of CYP1A1 gene and other genes

Other “inducers” also interact with receptors

• CAR, responds to phenobarbital-type inducers, regulates CYP2B, CYP3A4, CYP reductase, transferases (?)

• PXR, CYP3A• PPARα, CYP4A• LXR, FXR control enzymes involved in

bile acid and lipid metabolism

Phase II: Conjugation

• Synthetic reaction of a xenobiotic (or of a Phase I metabolite of a xenobiotic) with an endogenous substance

• Results in introduction of polar, ionizable groups to enhance water solubility and hence excretion

Major Phase II reactions

• Glucuronidation• Sulfation• Conjugation with amino acids• Conjugation with glutathione• Methylation• Acetylation

Glucuronidation• Enzyme: glucuronyl transferase, or

glucuronosyl transferase• Targets:

– hydroxyl groups: Phenols, Alcohols, Dihydrodiols (ether glucuronides)

– Carboxylic acids (ester glucuronides)– Amines (N-glucuronides)– Thiols (S-glucuronides)– Carbon (C-glucuronides, rare)

Reaction

OHo o

OH

HO

OH

COOH

PhenolPhenyl glucuronide

Glucuronidation

• Conjugating moiety: glucuronic acid, a sugar• Co-factor: UDP-glucuronic acid (UDPGA),

derived from glycogen synthesis• Located in endoplasmic reticulum• Multiple families of isoforms:UGT1, UGT2

– UGT1.1 ..1.7, UGT2.1..2.4• Inducible

Uridine-5’-diphospho--D-glucuronic acid (UDPGA)

O

OOH

OH

COO-

P

OH

OHO

OP

OH

O

OH

CH2O

OH

HN

N

O

O

O

GlucuronidationTypical substrates:

• Phenol• 1-Naphthol• 4-Hydroxybiphenyl• 3-Hydroxybenzo[a]pyrene• Benzo[a]pyrene-7,8-dihydrodiol• 2-Naphthylamine• Bilirubin• Steroids

Sulfation• Sulfotransferase ST,15 isoforms (xx-ST)• Targets

– Hydroxyl groups (phenols, alcohols)– Amino groups– Thiols

• Conjugating moiety: sulfuric acid, H2SO4

• Co-factor: 3’phosphoadenosine 5’phosphosulfate (PAPS), formed from ATP + sulfate

• Located in cytosol, Probably not inducible

SulfationTypical substrates

• Ethanol• Phenol• 3-Hydroxybenzo[a]pyrene• Cholesterol• 2-Naphthylamine• N-hydroxy-2-naphthylamine

Reaction

OH OSO3-

PAPS

PAP

Conjugation with amino acids

• Amino acid transferases• Targets: carboxylic acids• Conjugating moieties: Glycine, glutamine,

alanine, taurine, histidine, ornithine• Co-factor: Acetyl CoA (CoASH) and ATP• In cytosol

Reaction

C OH

O

CS CoA

OCoASCOCH3+ ATP

- AMPCH3COO-

CNH2

CH2 COO-

O+ NH2CH2COO-

-CoASH

Benzoic acid Benzoyl-CoA Hippuric acid

Conjugation with glutathione

• Glutathione S-transferases (GST)• Targets: Epoxides, halogens• Conjugating moiety: Glutathione• Co-factor: None• Mainly in cytosol• Inducible• Multiple families of isoforms: GSTA, GSTM,

GSTP, GSTT ()(αμπθ)

Glutathione

NH2CH

-OOCCH2

CH2

CNH CH

CH2

CNH

CH2COO-

SH

OO

Glutamic acid (Glu)

Glycine (Gly)

Cysteine (Cys)

A tripeptide

Reaction

OSG

HOH

+ GSH

H

Typical substrates

• Organic halides, e.g methyl iodide, benzyl chloride

• Alkenes e.g. diethyl maleate• Epoxides

Mercapturic acid pathway

C ClH

H H

C SH

+ GSH Glu

Cys

Gly

Gly

Cys

Glu

C S

H

H C S

H

H C

H

H-Cys

Glu

SCysNCOCH3H

Methylation

• Methyltransferases• Target: Hydroxyl groups, amines, thiols• Substrates mainly endogenous:

Catechols, noradrenalin, histamine• Conjugating moiety: Methyl group• Co-factor: S-adenosylmethionine

S-adenosylmethionine

N

N

NH2

O

OH

CH2S+

CH3

(CH2)2CH

-OOC

H2N

HO

MethylationReaction

OH

OH

OH

O CH3SAM

Substrate:

Catechol

Enzyme:

Catechol-O-methyltransferase (COMT)

Acetylation

• N-acetyltransferases (NAT)• Target: Aromatic amines, sulfonamides• Conjugating moiety: Acetyl group• Co-factor: Acetyl-CoA• Few forms: NAT1, NAT2. NAT3: mice• Genetic polymorphisms: “slow and fast

acetylators”

AcetylationReaction

NH2 N CH3

OH

Acetyl CoA

CoASH

C

2-Naphthylamine

2-Aminonaphthalene

2-Acetylaminonaphthalene

2-Acetamidonaphthalene

“Other” detoxication mechanisms

• P-glycoprotein: ATP-dependent carrier that removes molecules from cells

• Multidrug resistance associated protein MDR

• Multispecific organic anion transporter MOAT

Reactive Oxygen Species (ROS)

• Peroxides– Hydrogen peroxide HOOH– Peroxynitrite OONO-

– Lipid hydroperoxide LOOH• Free radicals

– Superoxide anion O2 •-

– Hydroxyl radical HO•

– Nitric oxide NO•

Non-enzymic reaction with anti-oxidants

• Ascorbic acid (Vitamin C)• alpha-Tocopherol (Vitamin E)• Glutathione

O

OHOH

O

C HHO

C H

OH

H

O CH3

CH3

H3C

HO

CH3

CH3 CH3 CH3CH3

Superoxide dismutase

Converts superoxide anions to hydrogen peroxide

O2•- + O2

•- + 2H+ O2 + H2O2

Peroxidases

Couple reduction of hydrogen peroxide (or other peroxide) to oxidation of another substrate

(co-oxidation)

ROOH + R’H ROH + R’OH

Peroxidases

• Catalase• Prostaglandin synthetase• Myeloperoxidase• Lactoperoxidase• Glutathione peroxidase

Glutathione peroxidase

GSH + GSH GSSG

HOOH HOH + HOH

Metabolic Activation/Metabolic Detoxication

• “Metabolism is a double-edged sword”• Generation of (re)active intermediates• Detoxication of (re)active intermediates

Pharmacologically active

Chemically reactive

Major reactive species

Electrophiles

Epoxides

(Epoxide hydrolaseGlutathione S-transferase)

Carbonium ions

Arylnitrenium ions

Reactive Oxygen Species