3. Metabolism Many xenobiotics undergo chemical transformation (biotransformation; metabolism) when introduced into biologic systems like the human body. Biotransformation is often mediated by enzymes End result of biotransformation is either alteration of the parent molecule, or conjugation of the parent molecule (or its metabolites) with endogenous substances in the body. Enzymes involved in biotransformation can act on either endogenous or xenobiotic compounds, especially if the xenobiotics are structurally similar to endogenous compounds

Example: Monoamine oxidase (MAO) is an enzyme that normally metabolizes biologic amines like epinephrine. MAO can also oxidize a variety of drugs. If a person is taking a drug that inhibits MAO activity (like many blood pressure medications), it can be dangerous for that person to take other drugs that can be metabolized by MAO.

Location of metabolic enzymes The products of biotransformation can be either less toxic, more toxic, or about as toxic as the parent molecules. Enzymes involved in biotransformation are sometimes called “drug metabolizing enzymes”. Although strictly speaking this is a misnomer because many of the substrates are not drugs, the term is still commonly used. Location of metabolic enzymes Species – found in virtually every species, although the type and amount vary tremendously. Organs – present in many tissues. Many enzymes are particularly abundant in the liver. Subcellular drug-metabolizing enzymes are located in the smooth endoplasmic reticulum (SER).

Types of Biotransformation Reactions Basically, two types of reactions, nonsynthetic (Phase I) and synthetic (Phase II) Phase I reactions Involve modification of the basic structure of the substrate Do not involve covalent binding of the substrate to an endogenous compound Phase I enzymes are often membrane-bound (e.g., microsomal). This is because they generally act on more lipid-soluble (nonpolar) substrates, and their purpose is to make the compounds MORE POLAR and therefore, MORE EASILY EXCRETABLE by the kidney and biliary tract.

Major Biotransformation Reactions Phase II Phase I Sulfation Oxidation Glucuronidation Reduction Glutathione conjugation Hydrolysis Acetylation Hydration Amino acid conjugation Dehalogenation

Oxidation Uses molecular oxygen (O2). One atom of oxygen is combined with hydrogen to form water, and the other atom of oxygen is introduced into the substrate molecule. Involves several enzymatic steps. The oxidative system is often known as the “mixed function oxidase” system”. These enzymes are some of the most thoroughly researched enzymes in biological systems. One subfamily of the mixed function oxidase system is the group of enzymes known as Cytochrome P-450 enzymes. They are so called because of their absorbance characteristics at wavelengths of 448-450 nm.

Anything that affects the activity of any oxidative enzyme can affect the way the body reacts to a given drug or other xenobiotic. Deamination – replacement of an amine group (NH2) with an oxygen (O) atom N-, O-, or S-Dealkylation – replacement of an alkyl group (e.g., CH3) with a hydrogen atom. Typically, the alkyl group in the parent molecule is bonded to a N, O, or S atom. Aliphatic or aromatic hydroxylation – addition of a hydroxyl group (OH) to a molecule N-oxidation – replacement of a hydrogen atom on an amine with an oxygen S-oxidation – addition of an oxygen atom to a sulfur atom Conversion of a hydroxyl group (alcohol) to a carboxyl group (acid)

Reduction Azo reduction – reduction of an azo bond (N=N) to two amines (NH2) Nitro reduction – reduction of a nitro group (NO2) to an amine Hydrolysis Addition of water (H2O) to an ester bond (CO-O-C) to form an alcohol (C-OH) and a carboxylic acid (COOH)

Phase II reactions Involve addition of a cofactor to a substrate to form a new product. Therefore, the rate of these reactions can be limited by the availability of the cofactor. Phase II enzymes may be either microsomal or cytosolic. This is because the primary purpose of the Phase II reactions is not so much to increase the polarity of the parent compound (although that is part of what they accomplish). The primary purpose is to increase the molecular weight of the parent compound to make it a better substrate for active transport mechanisms in the biliary tract. Various factors can affect the availability of cofactors. For example, fasting markedly reduces the amount of glutathione available in the liver.

Sulfation Replacement of a hydrogen atom (H) with a sulfonate (SO3-) Uses the enzyme sulfotransferase Uses the cofactor called PAPS (phosphoadenosine phosphosulfate) Produces a highly water-soluble sulfuric acid ester

Glucuronidation Replacement of a hydrogen atom with a glucuronic acid Uses the enzyme UDP-glucuronosyl transferase (UDP-GT) Uses the cofactor called UDPGA (uridine diphosphate glucuronic acid) One of the major Phase II enzymatic pathways Example: Conjugation of a phenol and a carboxylic acid with glucuronic acid

Acetylation Methylation Replacement of a hydrogen atom with an acetyl group Uses the enzyme acetyltransferase Uses the cofactor called acetyl CoA (acetyl coenzyme A) Sometimes results in a less water-soluble product Methylation Replacement of a hydrogen atom with a methyl group Uses the enzyme methyltransferase Uses the cofactor called SAM (S-adenosyl methionine) Common but relatively minor pathway

Glutathione conjugation Adds a glutathione molecule to the parent compound, either by direct addition or by replacement of an electrophilic substituent (e.g., a halogen atom) Uses the enzyme glutathione transferase (GST) Uses the cofactor called glutathione (a tripeptide made up of glycine, cysteine, and glutamic acid One of the major Phase II enzymatic pathways

Example: Metabolism of naphthalene showing the conjugation of naphthalene epoxide with glutathione and the subsequent formation of a N-acetylcysteine conjugate (mercapturic acid)

Amino acid conjugation Mercapturic acid formation Adds an amino acid to the parent compound. Mercapturic acid formation Formed by cleavage of the glycine and glutamic acid substituents from a glutathione conjugate, followed by N-acetylation of the resulting product

Significance of Biotransformation Reactions in Toxicology Biotransformation is a major part of the pathway for elimination of many xenobiotic compounds. Biotransformation can result in either a decrease or an increase (or no change) in toxicity. Biotransformation can result in the formation of reactive metabolites.

Example – metabolism of acetaminophen Acetaminophen is ordinarily metabolized in the liver by sulfation and glucuronidation to form non-toxic conjugates These are low capacity pathways, in that the cofactors are available in only limited concentrations, so these are rate-limiting. As long as the amount of acetaminophen in the liver is relatively low, the Phase II pathways can handle the compound, and there is no toxicity. If the concentration of acetaminophen becomes high enough to overwhelm the capacity of the Phase II pathways, an alternate metabolic pathway, involving Phase I enzymes, becomes active.

The product of the Phase I reaction is a highly reactive quinoneimine, which can form adducts with (bind covalently to) cellular macromolecules, especially proteins. The binding of the reactive intermediate to cellular macromolecules destroys the activity of those molecules, and can lead to compromised cell function and, ultimately, cell death.

Another good example – metabolism of carbon tetrachloride Carbon tetrachloride is metabolized by the cytochrome P-450 system in the liver by abstraction of one of the four chlorine atoms. This results in formation of a highly reactive trichloromethane radical, which initiates a cascade of lipid peroxidation by removing a hydrogen atom from membrane phospholipids. Damage to the cell membrane causes loss of osmotic integrity, cell swelling and death.

The activity of drug metabolizing enzymes is dependent on numerous factors Species Age (activity is generally lower in very young and aged animals) Sex (activity is generally higher in males than in females) Genetics (remember slow versus fast acetylators) Organ (activity of many enzymes is highest in the liver) General health status (e.g., hepatic injury decreases metabolic activity in the liver) Diet (remember how fasting decreases the amount of glutathione available for GST) Previous exposure to other compounds

Induction – an increase in the activity of one or more enzymes as a result of previous exposure of the organism to compounds that serve as substrates for the enzyme(s) Classic example of an inducer is phenobarbital, which induces the activity of cytochrome P-450 enzymes Induction may involve either increases in the synthesis of enzymatic protein, or increases in activation of proenzymes. One effect of induction of microsomal enzymes is an increase in the amount of smooth endoplasmic reticulum in a cell.

Induction is usually temporary, and enzyme activity levels return to normal after several weeks. Induction can result in tolerance to drugs, if the metabolism of the drugs results in a product with lower (or no) pharmacologic activity. This is why, for example, patients can develop tolerance to Phenobarbital anesthesia after repeated administration. Induction may result in increases or decreases in toxicity, depending on whether the metabolite is more or less toxic than the parent compound. This is why, for example, alcoholics are more susceptible to acetaminophen toxicity, since alcohol induces the enzyme that is responsible for production of the reactive metabolite from acetaminophen.

Inhibition – a decrease in the activity of one or more enzymes Classic example of an inhibitor is SKF-525A, which inhibits microsomal enzymes Inhibition may be either competitive or non-competitive. Competitive inhibition Occurs when an inhibitor binds to the same active site on the enzyme as the substrate. The higher the concentration of the inhibitor, the less likely it is that the substrate molecule will be able to find and bind to an available enzyme molecule. Reversible, since the binding of the inhibitor to the active site is not covalent Example: Omeprazole and diazepam are both metabolized by cytochrome P-450 2C19 (CYP2C19). Co-administration of these two drugs results in prolonged plasma half-life for diazepam.

Non-competitive inhibition May occur when an inhibitor binds to the same active site on the enzyme as the substrate, but binds so tightly that it is effectively not released. Thus, the binding site is permanently blocked. May also occur when an inhibitor binds tightly (sometimes covalently) to a different site on the enzyme than the active site. This can result in conformational or affinity changes that effectively inactive the enzyme. Non-competitive inhibition is generally not reversible. Therefore, recovery takes much longer because it requires the synthesis of new enzyme.

Factors affecting metabolism Age The metabolizing enzymes in neonates are not fully developed, therefore those cannot efficiently metabolize drugs. Also in the elderly, enzymatic systems may not function well leading to same conclusion. 2. Sex Males who are deficient in glucose -6-phosphate dehydrogenase are more prone to hemolysis when subjected to some drugs like sulfonamides

3. Pharmacogenetic factors Some individuals may be deficient in some enzymes, regardless of sex 4. Pregnancy Hepatic metabolism of drugs is decreased in pregnancy 5. Nutritional status/ liver dysfunction Malnutrition can cause a decreased level of some enzyme system and liver dysfunction can lead to decreased metabolism

6. Bioactivation Some drugs may be transformed to more toxic metabolites 7. Enzyme induction / inhibition A result of this is either an increase in the metabolism or a decrease in the drug metabolism 8. Changes in the kinetic mechanism: depending on whether the concentration of drug is in the therapeutic or overdose range