Transcript Metabolism of xenobiotics II
ENVR/TOXC 442 Fall 2012
Metabolism of Xenobiotics
II. Phase 1 Metabolism Aug 23, 2012 L.M. Ball Rosenau 158 [email protected]
Phase I reactions
• Chemical modification of xenobiotics • Introduces or uncovers polar functional groups that provide sites for Phase II metabolism • Major classes of reaction: – Oxidation – Reduction – Hydrolysis
Overview of oxidations, reductions, hydrolyses
•
Oxidation
–Loss of electrons M M + + e –Gain of oxygen R + O RO
Oxidation reactions
Hydroxylation
Epoxidation HC CH 2 HC O CH 2 Styrene Styrene oxide Benzo[
a
]pyrene H O H Benzo[
a
]pyrene 7,8-oxide
Demethylation CH 3 O OH Phenol + H O C H Formaldehyde Anisole Deethylation H 2 C CH 3 O N H C O Phenacetin CH 3 OH + H C o CH 3 N H C CH 3 O Acetaminophen Acetaldehyde
Overview of oxidations, reductions, hydrolyses
•
Reduction
– Gain of electrons M + + e M – Loss of oxygen RO R + O – Gain of hydrogen R + H RH
Reduction
• Nitro to amino group NO 2 NO H NOH H NH • Chromium VI to Chromium III Cr 6+ + 3 e Cr 3+
Hydrolysis
•
Addition of water
– Cleavage of R-O or R-N bond accompanied by addition of H 2 O R’-O-R + H 2 O R’-N-R + H 2 O H R’-O-H + R-OH R’-N-H + R-OH H
Principal Phase I enzymes
• Cytochrome P450 • Flavin monooxygenase • Monoamine oxidase • Esterases • Amidases • Hydrolases • Reductases, dehydrogenases, oxidases
Cytochrome P450
• Heme protein • Terminal oxidase of the mixed-function oxidase (MFO) electron-transfer system • Located in the smooth endoplasmic reticulum of all major organs and tissues • Uses NADPH as a source of reducing equivalents • Inducible
Cytochrome P450
• Heme protein • Terminal oxidase of the mixed-function oxidase (MFO) electron-transfer system • Located in the smooth endoplasmic reticulum of all major organs and tissues • Uses NADPH as a source of reducing equivalents • Inducible
Overall reaction
R-H + O 2 + NADPH + H + R-OH + H 2 O + NADP +
Ferric protoporphyrin IX
CH 3 HC CH 2 N OH O C H 2 C H 3 C H 2 C N Fe+3 N OH O C H 2 C CH 2 CH 3 N CH 3 CH CH 2
Protoporphyrin IX
Catalytic cycle of cytochrome P450
ROH H + Fe 3+ + RH NADH HO 2 2 Fe 3+ -RH H 2 O H + HO 2 [Fe 2+ -RH] H 2 O 2 O 2 [Fe 2+ -RH] +O 2 H + + e Fe 3+ -RH + e from NADPH-cytC reductase Fe 2+ -RH O 2
-.
NADPH
P450 and reductase in endoplasmic reticulum
The P450 gene superfamily
• Format of nomenclature: CYP
Family/Subfamily/Gene
• Family = 1, 2, …150 and counting – ~40% aa similarity • Subfamily = A, B,…H…W – 55-65% aa similarity • Gene = 1, 2..10 or above – >97% aa similarity (allelic variants) • Families grouped in Clans
Family Sub family
CYP1 (PAC-inducible A CYP2 A B C
Gene
1 BaP hydroxylation, O deethyl’n 2 N-hydroxylation, O-deethylation 1 Testosterone 7 -hydroxylation 2 Testosterone 15 -hydroxylation 1 Aliphatic hydroxylation 2 O-deethylation 1 - 20+ 2C19, mephenytoin hydroxylase
Demethylation CH 3 O OH Phenol + H O C H Formaldehyde Anisole Deethylation H 2 C CH 3 O N H C O Phenacetin CH 3 OH + H C o CH 3 N H C CH 3 O Acetaminophen Acetaldehyde
Family Sub family CYP1 (PAC-inducible A CYP2 A B C Gene 1 BaP hydroxylation, O deethyl’n 2 N-hydroxylation, O-deethylation 1 Testosterone 7 -hydroxylation 2 Testosterone 15 -hydroxylation 1 Aliphatic hydroxylation 2 O-deethylation 1 - 20+ 2C19, mephenytoin hydroxylase
Sub Family family CYP2 D CYP3 CYP4 E F A A Gene 1 - 6+ 2D6, debrisoquine hydroxylase 1 C- and N-hydroxylation small molecules 2 1 1-4 3A4 1 Lauric acid - and -1 hydroxylation
Polymorphisms
Sub Family family
CYP11 (mito) A
Gene
1 Steroid 11 -hydroxylation CYP17 CYP21 A A 1 Steroid 17 -hydroxylation 1 Steroid 21-hydroxylation CYP51 A CYP52-66 CYP71-99, 701 A CYP101 A CYP102-132 A 1 (Plants, yeast) Yeasts, fungi Plants 1
Pseudomonas putida
Bacteria P450 cam
Contributions of enzymes to the metabolism of Pfizer-marketed drugs.
Guengerich, 2008.
Changes in P450 levels with age Rats
M: 2C6, 2C11, 3A2 2A1 F: 2A1, 2C6, 2C12 2C6 3A2
• • • Protein data bank 101 Human P450 nomenclature Human P450s: substrates, inhibitors, inducers • Human CYP1A1 in Protein Knowledgebase
Flavin monooxygenase
• Flavoprotein • Mixed-function amine oxidase • Located in smooth endoplasmic reticulum, in human, pig, rabbit liver, guinea-pig lung, human kidney • Uses NADPH as a source of reducing equivalents • Not inducible
Overall reaction
R-H + O 2 + NADPH + H + R-OH + H 2 O + NADP +
Monoamine oxidase
• Metabolizes endogenous monoamine neurotransmitters • Uses NADPH as a source of reducing equivalents • Found in the endoplasmic reticulum and in mitochondria, of nerve endings and liver
Esterases
• Hydrolyse esters to carboxylic acid and alcohol functional groups • Non-specific esterases in plasma, more substrate-specific forms in liver cytosol H 3 C O C O H 2 C CH 3 Ethyl acetate + H 2 O O H 3 C C OH + HO H 2 C CH 3 Acetic acid Ethanol
Amidases
• Hydrolyse amides to carboxylic acids and amines (or ammonia) • Found in plasma and in liver cytosol R O C N H H + H 2 O R O C OH + H N H H
Hydrolases
• Hydrolyse ethers H 2 C H 2 C H 3 C O Diethyl ether CH 3 + H 2 O H 2 C H 3 C Ethanol OH H 2 C + HO Ethanol CH 3
Reductases, dehydrogenases, oxidases
• In cytosol, endoplasmic reticulum, mitochondria H 3 C H C
Alcohol dehydrogenase
OH NAD + NADH + H + H 3 C C
Aldehyde dehydrogenase
O H NAD + NADH + H + H 3 C C O OH H Ethanol Acetaldehyde Acetic acid
Outcomes
• Slight gain in water solubility (ionizable hydroxyl, amino groups) • Sites for Phase 2 reactions – attachment of glucuronyl, sulfate, glutathione residues > major gain in water-solubility.
• Formation of reactive electrophiles e.g. epoxides (especially polycyclic aromatic hydrocarbons) • Formation of sites for redox cycling (catechols <->quinones) > ROS