Biotransformation

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Biotransformation
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Results of Biotransformation

• Parent molecule becomes more polar
• Molecular weight and size incresed
• Excretion is facilitated
• Metabolic consequences
• Half life is decreased
• Duration of exposure reduced
• Accumulation in body reduced

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General points about biotransformation

• Enzymes involved are less specific
• Foreign compounds can enter endogenous pathways
• Foreign compounds can undergo more than one
  metabolic transformation
• Metabolism involves sequential steps(not always
  phase 1 then phase 2)
• Rates of various reactions can vary
• Biotransformation doesnt necessarily lead to
  detoxication

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Biotransformation

• Metabolic conversion of endogenous and xenobiotic
  nonpolar compounds to more polar or water-soluble
  compounds
• 2 groups of reactions
• Phase I
• Hydrolysis reactions
• Reduction reactions
• Oxidation reactions
• Phase II
• Conjugation reactions

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Enzyme Systems

• 2 major metabolizing systems
• Cytochrome P450 monooxygenase system (Phase I)
• Oxidation and some reduction reactions
• Conjugation or synthetic system
• Phase II
• Other enzymes
• Esterases
• Dehydrogenases
• Hydrolases
• Reductases

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Distribution of Enzymes

• Liver primary source
• Microsomes
• Endoplasmic reticulum
• Cytosolic
• Kidney
• Lung
• GI tract
• Eye

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Phase I Reactions

• Oxidative Reactions
• Microsomal
• Cytochrome P450 catalyzed reactions
• Flavin containing monooxygenase (FMO)
• Nonmicrosomal
• Alcohol and aldehyde dehydrogenases
• Monoamine and diamine oxidases
• Deamination
• Cooxidation
• Prostaglandin synthesis
• Hydrolysis
• Esterases

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Phase I Reactions

• Reduction Reactions
• Microsomal
• Cytochrome P450 catalyzed reactions
• Nonmicrosomal
• Glutathione reductases
• Disulfide reduction
• Ketone and aldehyde reductions
• Epoxide hydration

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Cytochrome P450 Monooxygenase System

• Located in the endoplasmic reticulum
• Primarily smooth ER
• Detoxifies/activates the highest number of
  xenobiotics
• Monooxygenation
• One atom of a molecule of oxygen is incorporated
  into the substrate
• Other atom reduced to water
• RH O2 NADPH H ? NADP ROH H2O

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Cytochrome P450

• Heme protein
• Many isoforms of the P450 enzymes
• Coupled enzyme system
• NADPH cytochrome P450 reductase
• NADH cytochrome b5 reductase
• NADPH is the hydrogen donor

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CP450 Catalytic Cycle

• Binding of substrate to oxidized P450
• One electron reduction of CP450 by NADPH CP450
  reductase
• Formation of ternary oxygenated complex
• One electron reduction by cytochrome B5 reductase
• Loss of one molecule of oxygen as water and
  transfer of one molecule of water to the
  substrate
• Release of oxidized substrate
• Return of CP450 to oxidized state

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CP450 Oxidation Reactions

• Epoxidation and aromatic hydroxylation
• Aliphatic hydroxylation
• Aliphatic epoxidation
• Heteroatom dealkylation
• Heteroatom oxygenation
• Oxidative deamination
• Desulfuration

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Flavin Monooxygenases

• FAD containing monooxygenase enzymes
• Oxidize nucleophilic nitrogen, sulfur and
  phosphorus atoms
• Microsomal
• Require NADPH and O2
• Similar to CP 450

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Oxidation Reactions

• Alcohol and aldehyde dehydrogenases
• Cytosolic enzymes
• RCH2OH NAD ?? RCHO NADH H
• Monoamine and diamine oxidases
• Oxidative deamination of primary, secondary and
  tertiary amines
• Located in brain and in mitochondria of liver,
  kidney and intestine
• Cooxidation
• Peroxidases
• Couples reduction of H2O2 to oxidation of other
  substance

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Hydrolysis Reactions

• Cytosolic and microsomal
• Hydrolysis of carboxylic acid esters, amides and
  thioesters
• Enzymes
• Carboxylesterases
• Acetylcholinesterases
• Pseudocholinesterases

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Hydrolysis Reactions

• Reactions
• Carboxylester hydrolysis
• RC(O)OR' ? RCOOH HOR'
• Carboxyamide hydrolysis
• RC(O)NR'R'' ? RCOOH HNR'RR''
• Carboxythioester hydrolysis
• RC(O)SR' ? RCOOH HSR'

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Hydrolysis Reactions

• Categorized by inhibition of specific compounds
• B esterases
• Inhibited by paraoxon (phosphotriester)
• Serine residue in active site
• A esterases Arylesterases
• Hydrolyze phosphotriesters
• C esterases Acetylesterases
• Prefer acetyl esters as substrates
• Phosphotriester neither substrate or inhibitor

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Reduction Reactions

• CP 450 reduction reactions
• Nitro reduction
• Azo reduction
• Quinone reduction
• Disulfide reduction
• Catalyzed by glutathione reductase
• Ketone and aldehyde reductions
• Reverse of alcohol dehydrogenases

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Epoxide Hydration

• Addition of water to alkene epoxides
• Enzyme
• Epoxide hydrolase
• Present in most tissues
• Cytosolic and microsomal
• Responsible for production of many toxic epoxides

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Phase II Reactions

• Conjugation reactions
• Cytosolic enzymes
• Exception glucuronosyl transferases
• Two types of reactions
• Type I Activated conjugating agent combines
  with substrate to yield conjugated product
• Type II Substrate activated and then combines
  with amino acid to yield conjugated product

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Conjugation Reactions

• Glucuronidation
• Sulfation
• Methylation
• Acetylation
• Amino acid conjugation
• Glutathione conjugation

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Glucuronide Conjugation

• Major Phase II reaction
• Enzyme
• Glucuronosyl transferase
• Microsomal
• Active donor
• UDP-glucuronic acid
• Glucuronic acid added to
• O, N, S, C

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Sulfate Conjugation

• Sulfate group added to hydroxyl groups present on
  phenols or aliphatic alcohols
• Enzyme
• Sulfotransferase
• Cytosolic
• Active donor
• PAPS
• Requires ATP and inorganic S

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Methylation

• Minor pathway
• May result in a DECREASE in water solubility
• Enzyme
• Methyl transferase
• Usually cytosolic
• Active donor
• S-adenosyl methionine (SAM)

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Methylation

• N-methylations
• Methylation of endogenous catechols
• O-methylations
• Microsomal enzyme
• Methylation of endogenous catechols
• S-methylations
• Cytosolic and microsomal
• Biomethylation
• Heavy metals

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Acetylation

• Acetylation of an amine group
• Enzyme
• N-acetyl transferase
• Cytosolic
• Active donor
• Coenzyme A

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Amino Acid Conjugation

• Type of acylation reaction
• Enzymes
• Mitochondrial or cytosolic
• ATP-dependent acidCoA ligases
• Acyl CoA synthetase
• Aminoacyl-tRNA synthetase

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Amino Acid Conjugation

• 2 pathways
• Conjugation of xenobiotic containing carboxylic
  acid group with the amino group of amino acids
• Activation of xenobiotic by conjugation with CoA,
  then reaction with amino group
• Conjugation of xenobiotic containing aromatic
  hydroxylamine with carboxylic acid group of amino
  acids

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Glutathione Conjugation

• Formation of N-acetylcysteine conjugates
• Enzymes
• Glutathione-S-transferases
• Cytosolic, liver
• Substrates
• Electrophilic!
• Conjugates may be converted to mercapturic acid
  in kidney and excreted

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Reactive Metabolites

• Two main types
• Reactive electrophiles
• Metabolic oxidation by CP 450
• Covalently bind to macromolecules, DNA
• Reactive nucleophiles
• Reduction with cofactor, NADPH
• Peroxidation of membrane lipids

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Activation Enzymes

• Oxidation
• Cytochrome P450
• Pulmonary prostaglandin synthetase
• Flavin-containing monooxygenase
• Alcohol and aldehyde dehydrogenases
• Xanthine oxidase
• Hydrolysis and reduction
• Cytochrome P450

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Suicidal Chemicals

• Inactivation of CP450 enzymes by active
  metabolite
• Competition for active site
• Destruction of enzyme
• Binding to heme
• Binding to protein structure
• Substrates
• Olefins, acetylenes
• Heteroatomic substrates

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Reactive Oxygen Species

• Reactive oxygen species
• Superoxide anion, hydrogen peroxide,
• Hydroxy radical, singlet oxygen
• Toxic effects
• Lipid peroxidation
• DNA strand breakage
• Attacks amino acids in active sites of enzymes

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Reactive Oxygen Species

• Generation of ROS
• Redox cycling
• Autooxidation reactions
• Formation of singlet oxygen via photochemical
  oxidations

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Common Reactive Metabolites

• Epoxides
• React with nucleophilic sites
• Deactivated by glutathione or conversion to
  dihydrodiol by epoxide hydrolase
• Quinones
• Autooxidation, Redox cycling
• Free radicals
• ROS
• Unstable conjugates

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Deactivation

• Glutathione
• Traps electrophilic metabolites
• Ascorbic acid, Vitamin E
• Antioxidants
• React with free radicals to form stable products
• Superoxide dismutase and catalase
• Reduce superoxide and hydrogen peroxide

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Modulation of Metabolism

• Primarily due to induction or inhibiton of the
  CP450 monooxygenase system
• Nutritional effects
• Decreased CP450 activity
• Low protein diet
• High carbohydrate diet
• Deficiency in linoleic acid or unsaturated fats
• Vitamin and mineral deficiencies

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Physiological Modulation of CP450

• Decreased CP450 monooxygenase activity associated
  with age
• Neonates
• Geriatric
• Hormonal
• Decreased activity
• Adrenal hormones
• Increased activity
• Thyroid
• Estrogen
• Decreased activity with disease

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Inhibition of CP450 by Chemicals

• Experimental demonstration
• In vivo symptoms
• Blood levels
• Metabolism of another xenobiotic in vivo
• In vitro metabolism following in vivo treatment
• In vitro metabolism

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Inhibition of CP450 by Chemicals

• Endogenous compounds
• Bile acids
• Hormones
• Acetyl coenzyme A
• Chemicals
• Promethazine
• Diazepam
• Organophosphates
• Heavy metals

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Induction of CP450 by Chemicals

• Two classes
• Phenobarbital
• Increase in proliferation of SER and amount of
  CP450
• Lower potency
• TCDD
• Increase in CP450 only
• Binds cytosolic receptor and increases
  transcription of CP450 genes

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Induction of CP450 by Chemicals

• Chemicals
• Carbamazepine
• Dexamethasone
• St. Johns Wort
• Ethanol
• Cigarette smoke