Metabolism of Chemicals

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Metabolism of Chemicals
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• Metabolism (biotransformation) of compounds is
  essential for survival of the organism.
• Accomplished by a limited number of enzymes with
  broad and overlapping substrate specificity.
• Many of the enzymes are constitutively expressed,
  but some require the presence of a drug or toxic
  compound to be induced enzyme induction.
• Primary aims of Metabolism
• - the parent molecule is transformed into a
  more polar metabolite, often by the addition of
  an ionizable group usually more H2O-soluble
  than the parent compound.
• - MW often increases.
• - Excretion, and therefore, elimination,
  facilitated.

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Consequences of Metabolism

• The biological t1/2 decreases.
• Exposure duration decreases.
• Compound does not accumulate in the body.
• But biological activity may change.
• Duration of biological activity may be affected.
• Although H2O-solubility and elimination are often
  increases, detoxification is not always the
  result.

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Kinetics of Metabolism

• First-Order Kinetics The metabolism of
  xenobiotics is catalyzed by enzymes, most of
  which obey Michaelis-Menten kinetics.
• v rate of xenobiotic metabolism
  VmaxC/Km C
• In most environmental situations, C ltlt Km.
  Thus, the above eq. reduces to v rate
  VmaxC/Km.
• This indicates that the rate is directly
  proportional to CFree.
• This means that a constant percent of the
  chemical is metabolized per unit time.

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Kinetics of Metabolism (Contd)

• Zero-Order Kinetics With a few agents, such as
  aspirin, ethanol, and phenytoin, the doses are
  very large. Thus, C gtgt Km and the velocity
  equation becomes
• v rate of xenobiotic metabolism
• VmaxC/C Vmax
• The enzyme is saturated by the CFree.
• The rate of metabolism remains constant over
  time.
• The means that a constant amount of the chemical
  is metabolized per unit time (e.g., alcohol DH).

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Metabolism has 2 (or 3) Phases

• Phase 1 and Phase II (and Phase III).
• Liver has highest concentration of enzymes that
  catalyze Phase I and Phase II reactions.
• But enzymes are also located in skin, lungs,
  nasal mucosa, eye, GIT, kidney, ovaries, plasma,
  and placenta.
• Phase III reactions are relatively minor
  further metabolism of GSH conjugates from Phase
  II reactions.

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Phase I Reactions and Metabolism

• Involves the addition of a functional group, such
  as OH, -NH2, -SH, -COOH.
• May also expose an existing functional group.
• Usually results in a small increases in H2O
  solubility.
• Prepares the compound for Phase II metabolism.
• Types of Reactions
• Oxidation, reduction, hydrolysis, hydration,
  dehalogenation.

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Prostaglandin H2 plays an important role in the
activation of xenobiotics to toxic or
tumorigenic metabolites, particularly in
extrahepatic tissues that are low in CYP450s.
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Oxidation

• Majority are catalyzed by membrane-bound
  mono-oxygenases in the SER known as microsomal
  enzymes.
• Others are found in the mitochondria and
  cytoplasm.
• Microsomal oxidations most are catalyzed by 1
  enzyme system Cytochrome P-450 mono-oxygenase
  system.
• -- Collection of isozymes, which use heme as
  prosthetic group.

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Oxidation (Contd)

• Cytochrome P450 mono-oxygenase associated with
  NAPDH cytochrome P450 reductase, which transfers
  2 e-, 1 at a time, to cytochrome P450 molecules.
• Some are inducible up to 2 orders of magnitude.
• Differences in the isozymes present may be due to
  differences between species, gender, age, and
  nutritional status.
• Over 200,000 chemicals are thought to be
  metabolized by P450s and play a role in over 90
  of all drugs in clinical use today.

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Metabolic Cycle

• Step 1 Binding of the substrate to the enzyme
  takes place when the Fe is in the oxidized Fe3
  state. Binding occurs on a site close to the Fe
  so that the substrate can interact with the O2
  when it binds Fe.
• Step 2 First e- reduction of the
  enzyme-substrate complex. The Fe is reduced to
  Fe2 state by e- transfer from NAPDH via cyt.
  P-450 reductase.

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Metabolic Cycle (Contd)

• Step 3 Addition of molecular O2, which binds
  Fe2.
• Step 4 Addition of a 2nd e- from NAPDH or from
  cyt. b5. The complex then rearranges with
  insertion of 1 atom of O into the substrate to
  yield the product (ROH). The other O atom is
  reduced to H2O, which is the other product.

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Main reaction can be written as
NADP
NADPH H
ROH H2O
RH O2
Reduced P-450
Oxidized P-450
Or, alternatively,
ROH H2O
RH O2
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Nomenclature

• Gene Families currently 27 families based on
  sequence homologies.
• Greater than 40 sequence homology same family
  CYP protein CYP gene family indicated by
  Arabic numeral of 1,2,3, etc.
• Subfamilies indicated by letter, gt55 sequence
  homology.
• Protein indicated by an Arabic number.

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CYP1A1 as an example

• P-450s have broad (low) substrate specificity gt
  1 CYP (e.g., CYP1A1) may be able to metabolize
  many different substrates.
• Substrate specificity overlaps gt 1 substrate may
  be metabolized to several different products by
  several different CYPs. 1 CYP can also
  metabolize a substrate to several different
  products.
• But there is significant stereoselectivity.

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CYP1A1 as an example (Contd)

• CYP1A subfamily and 2 proteins (CYP1A1 and
  CYP1A2) have been found in all classes of the
  animal kingdom.
• The majority of chemical carcinogens are
  substrates and/or inducers of CYP1A1.
• Levels of CYP1A1 and CYP1A2 are regulated by the
  Ah receptor, which binds planar PAHs and their
  derivatives. TCDD is the most potent inducer of
  CYP1A1.

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CYP1A1 as an example (Contd)

• Translocation of the Ah receptor-ligand complex
  to the nucleus gives rise to transcriptional
  activation of the CYP1 genes, increases in CYP1
  mRNA, and microsomal CYP1 enzymes.
• Ligand binding to the Ah receptor is also
  associated oncogene activation with initiation of
  the PKC cascade, which leads to cell
  proliferation (hyperplasia and tumor formation)
  via modulation of EGF interactions.

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CYP1A1 as an example (Contd)

• CYP1A1 is readily inducible in lung tissue
  following tobacco smoke inhalation.
• There is no discovered endogenous role the Ah
  receptor to date.

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Flavin-Containing Mono-Oxygenase System

• Found in the microsomal fraction of liver,
  kidneys and lungs.
• Catalyzes hydroxylation reactions at N, S, and P
  atoms, but not at C atoms.

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Flavin-Containing Mono-Oxygenase System (Contd)

• Examples of Types of Oxidation Reactions
• Aromatic hydroxylation generally proceed via
  the formation of an epoxide intermediate, which
  can be further metabolized to dihydrodiols, then
  to catechols.
• Aliphatic hydroxylation Unsaturated compounds
  proceed via epoxide formation. Aliphatic
  hydrocarbons are more likely to be metabolized if
  they are side-chains on aromatic structures.

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Flavin-Containing Mono-Oxygenase System (Contd)

• Alicyclic hydroxylation With mixed aromatic and
  alicyclic systems, hydroxylation of alicyclic
  systems predominates.
• Heterocyclic hydroxylation.
• N-, S-, O-Dealkylation.

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Reduction Reactions

• Located in both microsomal fraction and
  cytoplasm.
• Present in GIT microflora.
• Examples of Reductions
• -- Azo- and Nitro-Reductions
• -- Disulfide reduction
• -- Reductive dehalogenation
• -- Quinone Reduction

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Hydroylsis Reactions

• Enzymes include carboxlesterases, peptidases, and
  epoxide hydrolases.
• Acetylcholinesterase involve in organophosphorus
  insecticide metabolism.
• Epoxide hydrolase involved in converting epoxides
  to dihydrodiols.
• Epoxide hydrolase is found in ER in close
  proximity to P450 enzymes.

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Phase II Reactions and Metabolism

• Products from Phase I reactions and other
  xenobiotics containing functional groups OH, NH2,
  COOH, epoxide, or halogen, can undergo
  conjugation reactions with endogenous metabolites
  (e.g., sugars, aas, GSH, SO4, etc.
• Conjugation products are more polar, less toxic,
  and more readily excreted than their parent
  compounds are.

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• Most Phase II enzymes are cytosolic however,
  glucuronidation reactions are microsomal.
• Types of Reactions
• Glucuronidation
• Sulphate conjugation
• Acetylation
• Methylation
• Conjugation with GSH
• Conjugation with amino acids

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Glucuronidation

• Major type of reaction that involves a
  high-energy co-factor.
• Has wide cross-species prevalence, but not in
  cats.
• Co-factor uridine diphosphate-glucuronic acid
  (UDPGA) reaction is catalyzed by UDP
  glucuronisyltransferase enzymes (several
  isozymes).
• Glucuronic acid (GA) is transferred to OH and
  COOH groups plus N, S, and occasionally C atoms.
  Bonding is to the C1 on GA moiety.

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Glucuronidation (Contd)

• Glucuronide conjugates of compounds are polar and
  H2O-soluble. Therefore, elimination occurs
  either via urine or bile, depending on MW.
• The COOH moiety of GA is ionized at physiol. pH,
  and the compounds are recognized by urinary and
  biliary organic anion transporters, which enable
  conjugates to be secreted into urine or bile.
• Co-factor availability can limit the rate of
  glucuronidation of drugs administered in high
  doses (e.g., aspirin, acetominophen).

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Glucuronidation (Contd)

• Although glucuronidation generally decreases
  biol. activity of the drug, occasionally, it is
  increased.
• e.g., bioactivation of N-hydroxy-3-acetylaminoflu
  orine UDPGA ? hepatocarcinogen.
• Also, NSAIDS, hypolipidemic drugs, and
  anticonvulsants UDPGA ? carcinogenic,
  cytotoxic, and immunologic effects.
• Low rates of glucuronidation predispose neonates
  to jaundice and to toxic effects of some
  antibiotics.

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Sulphate Conjugation

• High-energy co-factor is 3-phosphoadenosyl-5-pho
  sphosulphate (PAPS), which is formed from ATP and
  inorganic sulphate ? energetically expensive for
  the cell 2 ATP to process.
• Conjugation reaction is catalyzed by
  sulphotransferases located in the cytosol of
  primarily the liver, GIT, mucosa, and kidneys,
• Sulphate conjugation involves the transfer of
  SO3- from PAPS to drug or toxic compound.
• Sulphate conjugates excreted mainly in urine.
• Need not undergo prior Phase I rxn (Table 6-4).

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Sulphate Conjugation (Contd)

• Those excreted in bile may be hydrolyzed by
  suphatases present in the GIT microflora, leading
  to contributing to enterohepatic circulation.
• The inorganic sulphate precursor of PAPs may
  become depleted when large amounts of a foreign
  compound conjugated with sulphate, such as
  paracetamol, are administered.
• Sulphate conjugation may increase toxicity in
  certain rare cases.

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Acetylation

• The activated co-factor Acetyl coenzyme A.
• Acetyltransferase found in the cytosol of
  hepatocytes, GIT mucosa and macrophages.
• Products of acetylation may be less H2O-soluble
  than the parent compound metabolites tend to
  precipitate out in the urine in kidney tubules,
  leading to nephron necrosis.
• N-acetylation is a major route of
  biotransformation for foreign compounds
  containing an aromatic amine (R-NH2) or a
  hydrazine group (R-NH-NH2).

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Methylation

• Methyl donor, S-adenosyl-methionine (SAM) is
  formed from met and ATP. Methyltransferase is
  mainly cytosolic, but can also be in ER. CH3
  group is transferred to an O, N, or S atom.
• This is a common, but usually minor, form of
  biotransformation.
• Metals can also be methylated. Hg and As can be
  single or di-methylated, while Se can be
  single,-double- or triple-methylated.
• Methylation tends to increase lipophilicity,
  thereby increasing the toxicity of some compounds.

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Conjugation with Glutathione (GSH)

• GSH is a tripeptide (glu-cys-gly)
• GSH is one of the most important cellular
  defenses against toxic compounds. Found in most
  cells, but especially abundant in liver.
• Cys provides SH group so that GSH will react as
  the thiolate ion, GS-.
• GSH conjugation may be an enzyme-catalyzed
  reaction (GSH-S- transferase) or simply a
  chemical reaction.

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Conjugation with Glutathione (GSH) (Contd)

• GSH can react with C, O, N, and S atoms.
• GSH conjugates can be excreted, usually in bile,
  rather than in urine.
• Or, the conjugate can be further metabolized by
  removal of glu and gly, followed by acetylation
  of the cys NH3 group to yield mercapturic acid.
• Resistance to certain toxic compounds is often
  assoc. with an over-expression of GSH
  S-transferase.

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Rhodanese

• Mitochondrial enzyme that converts CN- to a far
  less toxic metabolite, thiocyanate
• CN- S2O32- SCN- SO32-
• cyanide thiosulphate thiocyanate
  sulfite
• SO32- SO43-
• Alternatively, CN detoxification can be achieved
  using 4-dimethylaminophenol, which induces
  methemoglobinemia.

Sulfite oxidase
Mb
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Conjugation with Amino Acids

• 2 Pathways
• (1) Conjugation of xenobiotics containing a COOH
  group with the NH3 group of aa.
• Most commonly occurs with gly.
• But usually also occurs with gln, arg, taurine,
  and ornithine.
• Involves activation of xenobiotics with CoA prior
  to reaction with aa.
• Conjugates are usually excreted in the urine.

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Conjugation with Amino Acids (Contd)

• (2) Conjugation of xenobiotics containing an
  aromatic hydroxylamine with carboxylic acid group
  of aas, such as ser and pro.
• Amino acids are activated by amino-tRNA synthase.

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Phase I and Phase II Reactions

• Phase I Usually the rate-limiting step.
• Induction Compound incr gene expression of a
  metabolizing enzyme.
• - Dont confuse this with inhibition or
  antagonism!
• - Occurs with a wide variety of cpds (PAHs,
  drugs, insecticides, etc.)
• - Induce mono-oxygenases, but these cpds all
  share the following property organic and
  lipophillic.

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Induction (contd)

• Induction of CYP450s incr rate of
  biotransformation of xenobiotics (see Table 6-2).
• Induction can incr activation of procarcinogens
  to DNA-reactive metabolites.
• Can lead to pharmacokinetic tolerance by which
  larger drug doses must be administered to achieve
  the desired therapeutic levels(blood) because of
  incr drug biotransformation

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Phase I and Phase II Reactions (cont)

• Inhibition P450s may be inhibited in 3 ways
• Competitive inhibition by 2 different drugs for
  the same P450 enzyme.
• Noncompetitive 1 of the drugs binds covalently
  to P450.
• Competitive, but the inhibitor is not a substrate
  for the affected P450.

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Types of P450 Ligands

• Type I Broad class of drugs, pesticides, and
  other cpds that bind to CYP450s at a hydrophobic
  site near the heme so as to cause conformational
  changes, making the active sterically accessible.
• Type II Ligands that actually interact with the
  heme Fe
• Thus, such ligands must be small, usually
  diatomic, assoc with organic cpds with N atoms
  with sp2 or sp3 nonbonded e- that are sterically
  accessible, such as gases (O2, CO, NO, CN, CO2)

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An Example of Drug Interaction as a Result of
Metabolism