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 CH3

SAM

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

Non-polar(lipophilic)

Hydrophobic

Lipophobic Hydrophilic(Polar)

XENOBIOTIC

INTERMEDIATE METABOLITE

ELIMINATION

WATER-SOLUBLE METABOLITE

May be reactive/toxic

Can accumulate in tissues

Phase I MetabolismOxidation

Phase II MetabolismConjugation

Sol

ubili

ty in

lipi

ds

Sol

ubili

ty in

wat

er

Phase III (?)Transport into bile, urine

Transporters (Phase III)Organic Anion Transporters

OAT1

OAT2

OAT3

OAT4

Antiport (dicarboxylates eg αKG)

Pump into cell

Organic Anion Transporting Polypeptides

OATP1A1

OATP-A (1A2)

Antiport (eg GSH)

Pump into cell

Organic Cation Transporters

OCT1

OCT2

OCT3

Uniport

Proton

Multidrug Resistance Transporters

MRP1, MRP2

MRP4, MRP6

MDR1, MDR3

ATP pump

Glucurinide, glutathione, sulfate conjugates

Pump out of cell

Lethal Synthesis

Methylation of Mercury: Methylmercury CH3-Hg+

Dimethylmercury CH3-Hg-CH3

http://www.chm.bris.ac.uk/motm/dimethylmercury/dmmh.htm

Glutathione conjugation of 1,2-dihaloalkanesGSH + Br-CH2-CH2-Br GS-CH2-CH2-Br

Ethylene dibromide

Episulfonium ion CH2GS+ CH2

Major reactive species

ElectrophilesEpoxides

(Epoxide hydrolaseGlutathione S-transferase)

Carbonium ionsArylnitrenium ionsEpisulfonium ions

Reactive Oxygen Species

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

CH3CH3 CH3

CH3

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

OH O-Glucuronide

OOH

HH OH

S-GlutathioneHH OH

O

H

HO

HOH

Glutathione conjugate

The main purpose of this pilot study is to identify an optimal concentration of diesel exhaust which can be used to study the risks of cardiac changes in healthy older subjects. Results from this study may increase the understanding of how gaseous and particulate air pollutants (which causes the haze seen in some polluted cities) may adversely affect the functioning of the human cardiovascular and respiratory systems. This pilot research will study the responses to diesel exhaust in healthy older subjects with the specific GSTM1 and GSTP1 genotype. For more information, please visit our web site at www.epastudies.org or call Westat EPA Recruiting at 966-0604. Approved June 08, 2007 by the Committee on the Protection of the Rights of Human Subjects Biomedical Institutional Review Board. IRB # 07-0190 (GCRC #2579). This email is sponsored by: U.S. Environmental Protection Agency Human Studies Division located on the UNC-Chapel Hill campus. This email is sponsored by: U.S. Environmental Protection Agency Human Studies Division This email is sponsored by: CEMALB

Another exampleOrganophosphate Insecticides:

• Parathion

• Malathion

Parathion

Malathion

Hydrolysis enzymes

• Serum cholinesterase BChE

• Serum paraoxonase PON1

• Polymorphisms in PON1 – differential sensitivity

Heart diseaseAtherosclerosis Gulf War Syndrome

Effect is the outcome of interaction between susceptibility and

exposure

Target organs

• What makes a particular organ a target for toxicity / infection ?

• What makes a particular organ or species susceptible ?

Portal of entry to Blood to Target Organ

e.g. Intestine to hepatic portal vein to liver to vena cava to heart to lungs back to heart to aorta to rest of body

Location, location, location

Intestines

Hepatic portal veinLiver

Vena cava Aorta

Lungs

Gut flora

• Reductions– nitro to amine

• Hydrolyses– Cleavage of glucuronides

NO NOHNO2 NHH H

Reaction

OHo o

OH

HO

OH

COOH

Glucuronidation

Reaction

OHo o

O H

H O

O H

C O O H

De-glucuronidation-glucuronidase

Aglycone Conjugate

Enterohepatic recirculation(EHC)

Liver

Intestine

• Presence/absence of receptors– Estrogen receptors, Ah receptor

• Presence/absence of transporters/carriers– Resistance to chemotherapy

• Presence of repair mechanisms– DNA repair

• Balance of metabolic activation/detoxication

Factors affecting xenobiotic metabolism

• Intrinsic– Species, strain, gender, age, genotype

• Physiological status– Temperature, time of day, season, – Health status, disease, stress– Diet, nutritional status

• Related to exposure– Route of administration, frequency and size of

dose, co-exposures (induction, inhibition)

Genetic polymorphisms

• CYP2D6 Debrisoquine hydroxylation (poor and extensive metabolizers)

• Acetylation (fast and slow acetylators)

• GSTM null genotype

Changes in P450 levels with ageRats

2A1

2C6

3A2

M: 2C6, 2C11, 3A2

F: 2A1, 2C6, 2C12