Toxicology - Chapter 3 Biotransformation

Aliphatic hydroxylation

**direct attachment of an -OH Major category of oxidation reaction carried out by P450 Aliphatic means that the chain of carbons is open and not like an aromatic ring.

Properties to aid in diffusion across membrane

- Lipophilic - Neutrally charged - Small particle size

Oxidation Reactions Catalyzed by P450

- hydroxylation - dealkylation - epoxidation Oxidation reactions change hydrophobic substrates into more polar products, which can then be conjugated and eliminated through excretion in the urine..

Ester

A chemical compound derived from an acid (organic or inorganic) in which at least one -OH (hydroxyl) group is replaced by an -O-alkyl (alkoxy) group. Usually, esters are derived from substitution reaction of a carboxylic acid and an alcohol.

Endogenous opioids

A family of peptide transmitters that have been called the body's own narcotics. The three kinds are enkephalins, endorphins, and dynorphins.

Oxidation

A second mechanism of primary metabolism that either detoxifies or bioactivates into a more toxic compound.

Inhibitor

A substance that slows down or stops a chemical reaction. Examples of inhibitors include carbon monoxide, which competes with oxygen for binding to the heme site.

Choline Ester Hydrolysis

Active against choline esters, which are carboxyl esters in which the leaving group alcohol is the quaternary.

Acetylation

Addition of acetyl groups neutralizes the basic histones and promotes a looser, more open configuration. Often detoxicative, although acetylation may not lead to a more hydrophilic product and, in some cases, may yield a better substrate for P450 or other phase I enzymes. This mechanism is greatly reduced in dogs and foxes, and there is a common recessive allele of the NAT2 gene that produces slow acetylation in humans.

Aromatic hydroxylation

Addition of hydroxyl (OH) group to aromatic ring Aromatic hydroxylation reactions are implicated in bioactivation of carcinogens through the formation of these reactive epoxide intermediates. For example, benzo[a]pyrene-7,8-diol is a proximate carcinogen that can be further epoxidated to form benzo[a]pyrene-7,8-diol-9,10-epoxide, which is even more reactive with DNA than the parent molecule.

The Role of Metabolism by Gut Flora

Although it has long been recognized that metabolism of xenobiotics by the gut microbiome could play a significant role in the physiological effects of drugs and toxicants, the importance of this factor may well have been underestimated. This is a complicated question to address, however, as it involves not only the characterization of gut flora (which, of course, varies from individual to individual), but also the characterization of the metabolic activity of those organisms. Also, not only does the microbial community affect the metabolism of xenobiotics, but exposure to xenobiotics in turn affects the metabolic activities of the microbial community. Studies on this subject continue, though, and suggestions have been made that clinical manipulation of the gut microbiome may actually prove to be an effective tool in managing patients' response to medications.

Serine

Amino acid used for biosynthesis of proteins

Alcohol Dehydrogenase (ADH)

An enzyme that converts ethanol to acetaldehyde in the first step of alcohol oxidation. This enzyme converts small alcohols to aldehydes and is found in a variety of tissues such as liver, kidney, and lung. Individuals within the human population differ in which isozymes they express, and differences in the speed of ethanol metabolism between populations depend at least in part on which isozymes are expressed by individuals in that population. Forms of ADH also differ between tissues. Higher levels of class I ADH are located in the liver, which is where most ethanol metabolism takes place. A type of ADH known as class IV ADH is located in the gastrointestinal (GI) tract and may play some role in ethanol metabolism as well. This form of ADH may also be responsible for the increased risk of gastric cancer seen with heavy ethanol consumption, as it leads to the production of the suspected carcinogen acetaldehyde in the upper GI tract.

Epoxidation

An epoxide is a cyclic ether with a three-atom ring. This ring approximates an equilateral triangle, which makes it strained, and hence highly reactive, more so than other ethers. Epoxidation is the chemical reaction which converts the carbon-carbon double bond into oxiranes (epoxides), using a variety of reagents including air oxidation, hypochlorous acid, hydrogen peroxide, and organic peracid.

Prodrug

An inactive drug dosage form that is converted to an active metabolite by various biochemical reactions once it is inside the body.

Bilirubin

An orange-yellow pigment formed in the liver by the breakdown of hemoglobin and excreted in bile.

Monoamine Oxidase Inhibitors (MAOIs)

Antidepressant medications that increase the amount of monoamine neurotransmitter in synapses. Monoamine Oxidase is a critical enzyme in the brain. MAO activity increased with age is thought to be a contributor to Parkinson's disease, a neurodegenerative disease.

Detoxification

Biotransformation of parent drug results in less toxic compound Detoxification is most efficient when the parent drug is transformed into a large size, is hydrophilic and carries a charge.

Intoxication or Bioactivation

Biotransformation of parent drug results in more toxic compound

Hydrolysis

Breaking down complex molecules by the chemical addition of water "As an example of a compound that undergoes hydrolysis, consider methoprene, an insecticide used for mosquito and fly control. Methoprene possesses chemistry similar to that of a fatty acid alkyl ester (Figure 3.1). During hydrolysis, esters are split through the addition of a molecule of water to yield an acid and an alcohol. (This is the reverse of esterification, in which an acid and an alcohol react to produce an ester accompanied by water.) Hydrolysis proceeds more rapidly in alkaline conditions because it is actually the hydroxyl ion that attacks an electrophilic carbon in the reaction. When fed to cattle, methoprene does not accumulate because it is hydrolyzed to produce isopropanol and an aliphatic acid metabolite."

Reduction

Certain compounds are actually reduced instead of oxidized. They work as electron acceptors in a similar way as oxygen. Reduction occurs more in oxygen depleted environments. The reaction is catalyzed by the likes of P450 and others. The resulting amino acids can be oxidized to form a more toxic compound. Activation Reaction > Detoxification Reaction

Case Study: Glutathione Transferase and Pesticides

Chemical weed control with atrazine in maize is possible because the maize glutathione transferase efficiently detoxifies the pesticide. Atrazine is used in greater quantity than any other pesticide in the United States primarily for maize. Both reduced glutathione and glutathione S-transferase activity are increased in corn and sorghum crops by adding herbicide safeners such as dichlormid and flurazole to certain herbicides. These additives increase the margin of safety, or selectivity between killing the competing weed species and the crop plant. The mechanism of increasing glutathione conjugation is unknown, but the enhancement of glutathione S-transferase appears to be via induction of transcription from secondary genes.

Flavin Containing Monooxygenases

Class of monooxygenases. Known for N-oxidation of tertiary amines. They are important in S-oxidation reactions and in the desulfuration of phosphonates—those organophosphorus pesticides that possess one phosphorus-to-carbon bond. Stabilized by presence of NADPH.

Alkene Oxidation

Common reaction "One example of alkene oxidation is the epoxidation of the cyclodiene insecticide aldrin to the 6,7-epoxide product dieldrin (Figure 3.6). Another cyclodiene, chlordane, is metabolized to heptachlor epoxide. Dieldrin and chlordane were registered for many uses in agriculture, and chlordane was the principal termite-proofing agent used over the past 30 years. However, the registrations of these and most other cyclodiene insecticides have been canceled by the EPA due to persistence and suspected carcinogenicity."

Phase II reactions (Secondary Metabolism)

Conjugation step Products of phase I can be rendered highly polar by conjugation to carbohydrates, amino acids, or small peptides. These products are excreted more easily. Research in phase II biotransformation is complicated by the need to hydrolyze conjugates with enzymes, such as glucuronidase, peptidase, or sulfatase, in order to recover, identify, and measure the quantity of the phase I product that had been conjugated. In many insects, phase II reactions are very efficient because certain insecticides are oxidized and excreted primarily as conjugated metabolites. In addition to glucuronidation, glutathione conjugation, and acetylation, many other phase II reactions are known. These include conjugations of phase I products to sulfate, glucose, glycine, and other molecules catalyzed by the respective transferase enzymes.

Dimers vs. Heterodimers

Dimers - A molecule or molecular complex which consists of two identical molecules Heterodimers - enzymes with non identical subunits

Carboxyl Ester Hydrolysis

Diverse group, with more than 20 genes in mouse and multiple genes in rat and humans. Hydrolysis is a most important reaction of esters. Acidic hydrolysis of an ester gives a carboxylic acid and an alcohol. Basic hydrolysis of an ester gives a carboxylate salt and an alcohol. Two major forms of carboxyl ester hydrolysis in humans are CES1 and CES2. Genetic variations in these two can affect metabolism rate.

Hydrolases

Enzymes that catalyze hydrolysis reactions, often named for the substrate (e.g. nucleases, peptidases, amidases, lipases)

Serine hydrolases

Formed by peptidases, carboxylester hydrolases, and cholinester hydrolases "These enzymes have, as a catalytic site, a serine residue that reacts with the substrate to form a transiently alkylated enzyme as the ester bond of the substrate is cleaved. Serine hydrolase genes have been cloned, and the enzymes have been found to contain highly conserved regions of amino acid sequences, especially a Phe-Gly-Glu-Ser-Ala-Glu sequence that includes the reactive serine of the catalytic site."

Case Study: Inductions by 3-MC and Related Compounds

Induction by 3-MC and related compounds has been explained through a series of experiments. Investigations in mice led to the discovery of a soluble protein receptor, or transcription. Investigations in mice led to the discovery of a soluble protein receptor, or transcription factor, called the Ah receptor, which was lacking in homozygous mutant Ah− mice. Mice with the Ah receptor, which has a high affinity for TCDD, were susceptible to poisoning by TCDD, which must undergo bioactivation to produce toxicity. Ah− mice, however, were not responsive to TCDD. Further studies demonstrated that after binding of 3-MC or TCDD to the Ah receptor, the whole complex translocates to the nucleus, where interaction with DNA results in transcription of the appropriate P450 genes.

S- or N-oxidation

Leads to biotransformation of parent drug in to an equally or more toxic product.

Desulfuration

Most organophosphorothioate and organophosphorodithioate insecticides are propesticides that are biotransformed to much more toxic and reactive organophosphates via desulfuration. The lower toxicity parent compounds are used rather than the metabolites because the metabolites are much too toxic for practical handling and application. About 400 organophosphorothioate active ingredients are applied in agriculture and household pest control and in the control of mosquitoes.

Cofactor

Non-protein helpers that may be bound tightly to the enzyme as a permanent resident, or may bind loosely and reversibly along with the substrate. A nonprotein molecule or ion that is required for the proper functioning of an enzyme.

Phase I Reaction

Oxidation, reduction, or hydrolysis of parent compound. .

Malathion (Figure 3.2)

Pathways of metabolism of organophosphates leading to detoxification and intoxication. Both a general case and a specific case (malathion) are shown. "Malathion, as mentioned before, is a substrate for carboxylic ester hydrolases and in fact is primarily metabolized through the hydrolysis of its carboxyl ester group. The metabolic product of malathion is malaoxon, which is an even more reactive acetylcholinesterase inhibitor than malathion. Malaoxon possesses both a substrate carboxyl ester group and an inhibitor phosphorothionate group. Malathion hydrolysis proceeds linearly; however, malaoxon hydrolysis is progressively inhibited, resulting in a progressive decline of the rate of hydrolysis. Although malaoxon is unusual in serving as both substrate and inhibitor for carboxylic ester hydrolases, the general pathways of intoxicative and detoxicative biotransformation illustrated by malathion apply to most organophosphate pesticides (Figure 3.2). Toxicity from these compounds depends on the rates of bioactivation versus detoxification—both of which are affected by factors that alter metabolism.)"

Phase I vs Phase II Reactions

Phase I adds the necessary functional group and phase II attaches the larger molecule. However, it should be noted that for a given substrate, phase II conjugation does not necessarily follow a phase I reaction; nor does phase I metabolism always precede conjugation.

Glucuronidation

Phase II metabolism that adds a glucuronic acid to phenols, alcohols, and carboxylic acids. Conjugation of phase I products with the activated nucleoside diphosphate sugar uridine diphosphoglucuronic acid (UDPGA). Glucuronidation is naturally important in the conjugation of bilirubin, an endogenous compound produced when heme released from the hemoglobin of dead erythrocytes is oxidized in the spleen. The glucuronide conjugation occurs on an oxygen atom, resulting in carboxylic acid or ether products. However, N-, S-, and C-glucuronidation can also occur. While generally detoxicative, the formation of N-glucuronides of arylamines and N-hydroxy aryl amines may enhance bladder cancer by aiding transport of the carcinogen from the liver to the bladder, where the conjugate may undergo acid hydrolysis, thus releasing the carcinogen.

Paraoxonases

Present in mammalian liver and serum, (PON1, PON2, PON3) also catalyze organophosphates through an unknown process. Unlike serine hydrolysis, it contains nothing resembling the serine-containing conserved sequence of the serine hydrolases. Instead of a serine active site, this enzyme perhaps uses a cysteine residue.the shape of the active site in a paraoxonase is the mirror image of the active sites of acetylcholinesterase and chymotrypsin. Still, acetylcholinesterase and paraoxonase likely have active sites of similar size (but smaller than either chymotrypsin or carboxyl ester hydrolase).

Metabolites

Products of enzyme catalyzed chemical changes in the parent drug or toxicant

Glutathione Transferase

Reducing agent that can help reverse radical formation before damage is done to the cell. Many tissues are rich in glutathione. Glutathione transferases are dimers or heterodimers. A wide variety of biotransformation reactions are catalyzed by glutathione transferases. Glutathione transferases may also play a role in the detoxification of endogenous lipid and nucleic acid hydroperoxides produced by superoxide radical attack. Linoleic acid hydroperoxide is a good substrate for several glutathione transferases. Important for cancer research. In some cases, tumors become resistant to antineoplastic agents due to the high expression of glutathione transferases.

Dealkylation

Removal of a an alkyl group from sulfur, nitrogen or oxygen. "One example of this type of reaction is the dealkylation of chlordimeform to form N-demethyl chlordimeform, which is again N-dealkylated to form N,N-didemethyl chlordimeform (products that are successively more toxic; Figure 3.7). These reactions proceed through the formation of reactive oxygen-inserted intermediates that may be carcinogenic."

Conjugates

Side chains that, during metabolism, make drugs more water soluble and more easily excreted by the kidney.

Factors That Influence Metabolism

Species - qualitative and quantitative differences in enzymes between species AND between individuals of the same species. Age - Metabolism improves with age, however Xenobiotic metabolism declines with age. Some to take into account when creating medication for the elderly. Biological Sex - Gender differences in metabolism also exist in many species, but to this point, they have not been shown to play a significant role in xenobiotic handling in humans. Diet Environmental Factors

Polymorphism

The coexistence of two or more distinct forms in the same population.

Mutagenesis

The creation of a mutation or change in the nucleotide sequence of an organism's DNA.

Dehalogenation

The elimination of a halogen from a compound - formally a reduction. Resulting in activation, typically and causes the formation and release of free radicals and other reactive intermediates with the strong potential to bind to cellular macromolecules. Halogens can be removed from compounds such as carbon tetrachloride by replacing them with hydrogen or oxygen or through elimination of two adjacent halogens and replacement with a double bond. Reduction can reverse oxidation and the two process can yo-yo back and forth which can actually decrease the elimination half life of a compound.

Acetylcholinesterase

The enzyme that breaks down acetylcholine in the synaptic cleft. "Critical enzyme for clearing the neuromuscular synapse of the neurotransmitter, acetylcholine."

Glutathione conjugation

The epoxide of aflatoxin B1 formed by P450-catalyzed oxidation can be conjugated with glutathione at a slow rate, and induction of higher rates of conjugation decreases the oncogenicity. In contrast, 1,2-dibromoethane (ethylene dibromide), a once common agricultural fumigant canceled by the EPA, is biotransformed to a highly carcinogenic glutathione conjugate that acts as a sulfur mustard to alkylate DNA. This activation is probably responsible for the high rate and rapid formation of stomach and nasal carcinoma observed in rats administered 1,2-dibromoethane.

Phase II Reaction

The formation of a metabolite conjugated (bound) to a larger molecule that is hydrophilic and carries a charge.

Cytochrome P450

The general name for a large class of enzymes that play a significant role in drug metabolism and drug interactions. Many P450 enzymes undergo the process of induction whereby enzyme activity increases following exposure to substrates. Phenobarbital is an inducing agent of CYP 2 and CYP3 families. 3-methylcholanthrene (3-MC) and tetrachlorodibenzodioxin (TCDD) are inducing agents of the CYP1 family. P450 activity can be inhibited. Not only can inhibitors of protein synthesis block the induction process, but also some compounds can act as specific competitive inhibitors for binding to the P450 enzyme. Examples of inhibitors include carbon monoxide, which competes with oxygen for binding to the heme site.

Case Study: Glutathione Transferase and Acetaminophen

The significant role of glutathione transferases in xenobiotic detoxication can be observed in the biotransformation of the common analgesic drug acetaminophen, which is transformed to N-acetylp-benzoquinonimine, a potential hepatotoxin, in a phase I N-oxidation. This reactive product is rapidly conjugated to glutathione in a reaction that is catalyzed readily by glutathione transferases. Depletion of glutathione by acetaminophen overdose negates this detoxication and can lead to liver toxicity or death in extreme cases.

Metabolomics

The study of the complete complement of small chemicals present in a cell at any given time "Advances in urinalysis with high-resolution nuclear magnetic resonance (NMR) have also introduced the potential to relate metabolism to genetics using urinary profiles as a phenotype. This topic is known as metabolomics or metabonomics. Generally, these methods consider the alteration of the normal profile of bodily metabolites, but could include the profile of xenobiotics if detected."

Cytosolic Classes (Glutathione transferases)

There are now, seven known classes of these enzymes: alpha, mu, pi, theta, kappa, sigma, and zeta. Enzymes within a class have subunits that share a much greater homology (around 70%) than the subunits of enzymes in different classes. Because all reactions occur even without catalysis, it appears that the glutathione sulfhydryl group is the active site and that the enzyme holds it in a position that enhances the nucleophilic attack on the substrate (which is typically electrophilic) and thus enhances the rate of transition to products. A wide variety of biotransformation reactions are catalyzed by glutathione transferases.

Epoxide Hydrolase

This enzyme converts an epoxide into a diol via addition of water. Located in cytosol. This helps avoid potentially toxic effects by epoxides.

in situ

in its original place

Aldehyde Dehydrogenase (ALDH)

the enzyme that catalyzes the conversion of acetaldehyde to acetate, which forms acetyl CoA. Acetyl CoA - Its main function is to deliver the acetyl group to the citric acid cycle (Krebs cycle) to be oxidized for energy production. There are at least 19 ALDH isozymes occurring in various human tissues, some of which show polymorphisms that may vary between individuals. Some individuals, for example, have a form of ALDH that works more slowly than other forms. If this variation happens to occur in conjunction with a variant form of ADH that produces an increased rate of conversion of alcohol to aldehyde, then aldehyde levels can build and cause unpleasant physiological symptoms such as flushing of the skin. Genetic variations in these enzymes are, in fact, considered to be one of the many potential factors that must be considered in attempting to assess the risk for alcoholism.