13: Biochemical and Metabolic Principles

Xenobiotics are compounds that are foreign to a living system. Toxic xenobiotics interfere with critical metabolic processes, causing structural damage to cells or altering their cellular genetic material. The specific biochemical sites of actions that disrupt metabolic processes are well characterized for many xenobiotics although mechanisms of cellular injury are not. This chapter reviews the biochemical principles that are relevant to an understanding of the damaging effects of toxic xenobiotics and the biotransformation enzymes and their clinical implications.

The capacity of a xenobiotic to produce injury is affected by many factors, including its absorption, distribution, elimination, site of activation or detoxification, and site of action. This section focuses on how the route of exposure and the ability to cross membranes in order to access particular organs affect toxicity.

The route of exposure to a xenobiotic may confine damage primarily to one organ, for example, pulmonary injury that follows inhalation of an irritant gas or gastrointestinal (GI) injury that follows ingestion of a caustic. Hepatocellular injury results when a toxic xenobiotic is delivered to the liver, either by the portal venous system following ingestion or by the hepatic artery carrying blood containing xenobiotics absorbed from other sites of exposure.

Various factors affect the ability of a xenobiotic to access a particular organ. For example, many potentially toxic xenobiotics fail to produce central nervous system (CNS) injury because they cannot cross the blood–brain barrier. The negligible CNS effects of the mercuric salts when compared with organic mercury compounds are related to their relative inability to penetrate the CNS. Two potent biologic xenobiotics—ricin (from Ricinus communis) and α-amanitin (from Amanita phalloides)—block protein synthesis through the inhibition of RNA polymerase. However, they result in different clinical effects because they access different tissues. Ricin has a special binding protein that enables it to gain access to the endoplasmic reticulum in GI mucosal cells, where it inhibits cellular protein synthesis and causes severe diarrhea. α-Amanitin is transported into hepatocytes by bile salt transport systems, where inhibition of protein synthesis results in cell death. The electrical charge on a xenobiotic also affects its ability to enter a cell. Unlike the ionized (charged) form of a xenobiotic, the uncharged form is often lipophilic and passes through lipid cell membranes to enter the cells. The pK_a of an acidic xenobiotic (HA↔A⁻ + H⁺) is the pH at which 50% of the molecules are charged (A^– form) and 50% is uncharged (HA form). A xenobiotic with a low pK_a is more likely to be absorbed in an acidic environment where the uncharged form predominates.

The capability to detoxify and eliminate both endogenous toxins and xenobiotics is crucial to the maintenance of physiologic homeostasis. A simple example is the detoxification of cyanide, a potent cellular poison that is common in the environment and is also a product of normal metabolism. Mammals have evolved the enzyme rhodanese, which combines cyanide with thiosulfate to create the less toxic, renally excreted compound thiocyanate.⁵

Most xenobiotics have lipophilic properties that facilitate absorption across cell membranes of organs that are portals of entry into the body: the skin, GI tract, and lungs. The liver has the highest concentration of enzymes that metabolize xenobiotics. Enzymes found in the cytosol of hepatocytes that are specific for alcohols, aldehydes, esters, or amines act on many different substrates within these broad chemical classes. Enzymes that act on more lipophilic xenobiotics, including the cytochrome P450 (CYP) enzymes, are embedded in the lipid membranes of the cytosol-based endoplasmic reticulum. Microsomes are the pieces of the endoplasmic reticulum that result when cells are disrupted. When cells are mechanically disrupted and centrifuged, these membrane-bound enzymes are found in the pellet, or microsomal fraction, hence they are called microsomal enzymes. Enzymes located in the liquid matrix of cells are called cytosolic enzymes and are found in the supernatant when disrupted cells are centrifuged.¹⁸

The study of xenobiotic metabolism was established as a scientific discipline by the seminal publication of Williams in 1949.⁹³ Biotransformation is the physiochemical alteration of a xenobiotic, usually as a result of enzyme action. Most definitions also include that this action converts lipophilic substances into more polar, excretable substances.^52,83 Most xenobiotics undergo some biotransformation, the degree of which is affected by their chemical nature. The hydrophilic nature of ionized compounds such as carboxylic acids enables the kidneys to rapidly eliminate them. Very ­volatile compounds, such as enflurane, are expelled promptly via the lungs. Neither of these groups of xenobiotics undergo significant enzymatic metabolism.

Biotransformation usually results in “detoxification,” a reduction in the toxicity, by the conversion to hydrophilic metabolites of the xenobiotic that can be renally eliminated.⁵² However, this is not always the case. Many parent xenobiotics are inactive and must undergo “metabolic activation,” a classic concept introduced in 1947.⁵⁴ When metabolites are more toxic than the parent xenobiotic, biotransformation has resulted in “toxification.”⁸³ Biotransformation via acetylation or methylation enhances the lipophilicity of a xenobiotic. Biotransformation is done by impressively few enzymes, reflecting broad substrate specificity. The predominant pathway for the biotransformation of an individual xenobiotic is determined by many factors, including the availability of cofactors, changes in the concentration of the enzyme caused by induction, and the presence of inhibitors. The predominant pathway is also affected by the rate of substrate metabolism, reflected by the K_m (Michaelis-Menten dissociation constant) of the biotransformation enzyme⁸³ (Chap. 9).

Biotransformation is often divided into phase I and phase II reactions, terminology first introduced in 1959.⁹⁴ Phase I reactions prepare lipophilic xenobiotics for the addition of functional groups or actually add the groups, converting them into more chemically reactive metabolites. This is usually followed by phase II synthetic reactions that conjugate the reactive products of phase I with other molecules that render them more water soluble, further detoxifying the xenobiotics and facilitating renal elimination. However, since biotransformation often does not follow this stepwise process, it has been suggested that phase I and II terminology be eliminated.³⁹ Some xenobiotics undergo only a phase I or a phase II reaction prior to elimination. Additionally, phase II reactions can precede phase I. While virtually all phase II synthesis reactions cause inactivation, a classic exception is fluoroacetate being metabolized to fluorocitrate, a potent inhibitor of the tricarboxylic acid cycle (Chap. 115).⁶⁹

Biotransformed xenobiotics cannot be eliminated until they are moved back across cell membranes, out of the cells. Membrane transporters are proteins that move agents across the membranes without altering their chemical compositions, a process called a phase III reaction because it typically occurs after biotransformation.³⁹ However, membrane transport does not always occur after phase I or II reactions. Some parent compounds are transported across membranes without any biotransformation at all.

Phase I Biotransformation Reactions

Oxidation is the predominant phase I reaction, adding reactive functional groups suitable for synthetic conjugation during phase II. These groups include hydroxyl (–OH), sulfhydryl (–SH), amino (–NH₂), aldehyde (–CHO), or carboxyl (–COOH) moieties. Noncarbon elements such as nitrogen, sulfur, and phosphorus are also oxidized in phase I reactions. Other phase I reactions include hydrolysis (the splitting of a large molecule by the addition of water that is divided among the two products), hydration (incorporation of water into a complex molecule), hydroxylation (the attachment of –OH groups to carbon atoms), reduction, dehalogenation, dehydrogenation, and dealkylation.^52,83

The CYP enzymes are the most numerous and important of the phase I enzymes. A common oxidation reaction catalyzed by CYP enzymes is illustrated by the hydroxylation of a xenobiotic R–H to R–OH (Fig. 13–1).²⁴ Membrane-bound flavin monooxygenase (FMO), an NADPH-dependent oxidase located in the endoplasmic reticulum, is an important oxidizer of amines and other compounds containing nitrogen, sulfur, or phosphorus.⁵²

The alcohol, aldehyde, and ketone oxidation systems use predominantly cytosolic enzymes that catalyze these reactions using NADH/NAD⁺ (Fig. 13–2).^49,83 Two classic phase I oxidation reactions are the metabolism of ethanol to acetaldehyde by alcohol dehydrogenase (ADH) followed by the metabolism of acetaldehyde to acetic acid by aldehyde dehydrogenase (ALDH). Alcohol dehydrogenase, which oxidizes many different alcohols, is found in the liver, lungs, kidneys, and gastric mucosa.⁴⁹ Women have less ADH in their gastric mucosa than men. This results in decreased first-pass metabolism of alcohol and increased alcohol absorption. Some populations, particularly Asians, are deficient in ALDH, resulting in increased acetaldehyde concentrations and symptoms of the acetaldehyde syndrome⁴⁹ (Chap. 79).

Oxidation Overview

Biotransformation often results in the oxidation or reduction of carbon. A substrate is oxidized when it transfers electrons to an electron-seeking (electrophilic or oxidizing) molecule, leading to reduction of the electrophilic molecule. These oxidation-reduction reactions are usually coupled to the cyclical oxidation and reduction of a cofactor, such as the pyridine nucleotides, nicotinamide adenine dinucleotide (NADH/NAD⁺), or nicotinamide adenine dinucleotide phosphate (NADPH/NADP⁺). The nucleotides alternate between their reduced (NADPH, NADH) and oxidized (NADP⁺, NAD⁺) forms. Because xenobiotic oxidation is the most common phase I reaction, the newly created reduced cofactors must have a place to unload their electrons; otherwise, biotransformation ends. The electron transport chain serves as the major electron recipient.

Electrons resulting from the catabolism of energy sources are extracted primarily by NAD⁺, forming NADH. Within the mitochondria, NADH transports its electrons to the cytochrome-mediated electron transport chain. This results in the production of adenosine triphosphate (ATP), the reduction of molecular oxygen to water, and the regeneration of NAD⁺—all parts critical to the maintenance of oxidative metabolism. NADPH, created within the hexose monophosphate shunt, is used in the synthetic (anabolic) reactions of biosynthesis (especially fatty acid synthesis). NADPH is also the cofactor in the reduction of glutathione, a molecule vital to the protection of cells from oxidative damage.

The oxidation state of a specific carbon atom is determined by counting the number of hydrogen and carbon atoms to which it is connected. The more reduced a carbon, the higher the number of connections. For example, the carbon in methanol (CH₃OH) has three carbon–hydrogen bonds and is more reduced than the carbon in formaldehyde (H₂C=O), which has two. Carbon–carbon double bonds count as only one connection.

Cytochrome Enzymes—An Overview

Cytochromes are a class of hemoprotein enzymes whose function is electron transfer, using a cyclical transfer of electrons between oxidized (Fe³⁺) or reduced (Fe²⁺) forms of iron. One type of cytochrome is cytochrome P450 (CYP) whose nomenclature derives from the spectrophotometric characteristics of its associated heme molecule. When bound to carbon monoxide, the maximal absorption spectrum of the reduced CYP (Fe²⁺) enzyme occurs at 450 nm.⁵⁹ CYP enzymes split the two oxygen atoms of an oxygen molecule, incorporating one into the substrate and one into water and thus are called mixed-function oxidases or monooxygenases. This differs from dioxygenases that incorporate both oxygen atoms into the substrate.^59,83

Cytochrome enzymes perform many functions. The biotransformation CYP enzymes are bound to the lipid membranes of the smooth endoplasmic reticulum. They execute 75% of all xenobiotic metabolism and most phase I oxidative biotransformations of xenobiotics.³² A second role for CYP enzymes is synthetic: biotransforming endobiotics (chemicals endogenous to the body) to cholesterol, steroids, bile acids, fatty acids, prostaglandins, and other important lipids. Cytochromes also act as electron transfer agents within the mitochondrial electron transport chain.^33,59

Although more than 6000 CYP genes exist in nature, the human genome project determined that the number of human CYP genes at 57.⁵⁹ CYP enzymes are categorized according to the similarities of their amino acid sequences. They are in the same “family” if they are more than 40% comparable and same “subfamily” if they are more than 55% similar. Families are designated by an Arabic numeral (n), subfamilies by a capital letter (X), and each individual enzyme by another numeral (m), resulting in the nomenclature CYPnXm for each enzyme. For example, CYP3A4 is enzyme number 4 of the CYP3 family and of the CYP3A subfamily.⁵⁶ Most xenobiotic metabolism is done by the CYP1, CYP2, and CYP3 families, with a small amount done by the CYP4 family.^10,90 Although 15 CYP enzymes metabolize xenobiotics,⁶⁶ nearly 90% is done by 6 CYP enzymes: 1A2, 2C9, 2C19, 2D6, 2E1, and 3A4 (Table 13–1).⁵⁹

Most CYP enzymes are found in the liver, where they comprise 2% of total microsomal protein.⁶⁶ High concentrations are also found in extrahepatic tissues, particularly the GI tract and kidney.^20,60 The lungs,⁹⁶ heart,⁶⁴ and brain²¹ have the next highest amounts. Each tissue has a unique profile of CYP enzymes that determines its sensitivity to different xenobiotics.²⁰ The CYP enzymes in the enterocytes of the small intestine actually contribute significantly to “first-pass” metabolism of some xenobiotics.^42,59 Corrected for tissue mass, the CYP enzyme system in the kidneys is as active as that in the liver. The activity of the renal CYP enzymes is decreased in patients with chronic kidney disease, with relative sparing of CYPs 1A2, 2C19, and 2D6 compared with 3A4 and 2C9.¹⁰

Cytochrome P450 Enzyme Specificity for Substrates

In vitro models are used to define the specificities of CYP enzymes for their substrates and inhibitors. However, activity in a test tube does not always correlate with that in a cell. These models use substrate and inhibitor concentrations that are much higher than would be encountered in vivo, and the mathematical models that extrapolate to clinically relevant processes yield conflicting results. This has resulted in discrepancies in reported substrates, inhibitors, and inducers of specific CYP enzymes.⁹²

Most CYP enzymes involved in xenobiotic biotransformation have broad substrate specificity and can metabolize many xenobiotics.³² This is fortunate because the number of xenobiotic substrates may exceed 200,000 and continues to grow.⁴⁸ Broad substrate specificity often results in multiple CYP enzymes being able to biotransform a xenobiotic. This enables the ongoing biotransformation despite an inhibition or deficiency of an enzyme. When a substrate can be biotransformed by more than one enzyme, the enzyme with the highest affinity for the substrate usually predominates at low substrate concentrations, whereas enzymes with lower affinity may be very important at high concentrations. This transition is usually concomitant with, but not dependent on, the saturation of the catalytic capacity of the primary enzyme as it reaches its maximum rate of activity.³² The K_m, which is defined as the concentration of substrate that results in 50% of maximal enzyme activity, describes this property of enzymes. For example, ADH in the liver has a very low K_m for ethanol, making it the primary metabolic enzyme for ethanol when concentrations are low.⁴⁹ Ethanol is also biotransformed by the CYP2E1 enzyme, which has a high K_m for ethanol and only functions when ethanol concentrations are high. The CYP2E1 enzyme metabolizes little ethanol in moderate drinkers but accounts for significantly more biotransformation in alcoholics. As another example, diazepam is metabolized by both CYP2C19 and CYP3A4 enzymes. However, the affinity of CYP3A4 for diazepam is so low (ie, the K_m is high) that most diazepam is metabolized by CYP2C19.³⁴

The substrate selectivity of some CYP enzymes is determined by molecular and physicochemical properties of the substrates. The CYP1A subfamily has greater specificity for planar polyaromatic substrates such as benzo[a]pyrene. The CYP2E enzyme subfamily targets low-molecular-weight hydrophilic xenobiotics, whereas the CYP3A4 enzyme has increased affinity for lipophilic compounds. Substrates of CYP2C9 are usually weakly acidic, whereas those of CYP2D6 are more basic.⁴⁸ High specificity can also result from key structural considerations such as stereoselectivity. Some xenobiotics are racemic mixtures of two stereoisomers that are substrates for different CYP enzymes and have distinct affinities for the enzymes, resulting in different rates of metabolism. For example, R-warfarin is biotransformed by CYP3A4 and CYP1A2, whereas S-warfarin is metabolized by CYP2C9.^76,83

The CYP enzymes that biotransform a specific xenobiotic cannot be predicted by its drug class. For example, fluoxetine and paroxetine are both major substrates and potent inhibitors of CYP2D6, but sertraline is not extensively metabolized by this enzyme and exhibits minimal interaction with other antidepressants.⁴ Most β-hydroxy-β-methylglutarylcoenzyme A (HMG-CoA) reductase inhibitors are metabolized by CYP3A4 (lovastatin, simvastatin, and atorvastatin); however, fluvastatin is metabolized by CYP2D6 and pravastatin undergoes virtually no CYP enzyme metabolism at all.⁵¹ Among angiotensin-II receptor blockers, losartan and irbesartan are metabolized by CYP2C9, whereas valsartan, eprosartan, and candesartan are not substrates for any CYP enzyme. In addition, losartan is a prodrug whose active metabolite provides most of the pharmacologic activity, whereas irbesartan is the primary active compound. For these two drugs the inhibition of CYP2C9 is predicted to have opposite effects.²⁶

Cytochrome P450 and Drug–Drug/Drug–Chemical Interactions

Adverse reactions to medications and drug–drug interactions are common causes of morbidity and mortality, the risk of which increases with the number of drugs taken (Chaps. 139 and 140). Fifty percent of adverse reactions may be related to pharmacogenetic factors.²⁸ The most significant interactions are mediated by CYP enzymes.⁵⁹ The impacts of genetic polymorphism and enzyme induction or inhibition are addressed below.

CYP enzymes are involved in many types of drug interactions. The ability of potential new drugs to induce or inhibit enzymes is an important consideration of industry. Drug development focuses on the potential of new xenobiotics to induce or inhibit other drugs or enzymes during the drug discovery phase. Various in vitro models have been created to enable this early determination.⁵⁰

Many xenobiotics interact with the CYP enzymes. St. John’s wort, an herb marketed as a natural antidepressant, induces multiple CYP enzymes, including 1A2, 2C9, and 3A4. The induction of CYP3A4 by St. John’s wort is associated with a 57% decrease in effective serum concentrations of indinavir when given concomitantly.⁶⁷ Xenobiotics contained in grapefruit juice, such as naringin and furanocoumarins, are both substrates and inhibitors of CYP3A4. They inhibit the first-pass metabolism of CYP3A4 substrates by inhibiting CYP3A4 activity in both the GI tract and the liver.¹⁷ Polycyclic hydrocarbons found in charbroiled meats and in cigarette smoke induce CYP1A2. Thus, for smokers who drink coffee, concentrations of caffeine, a CYP1A2 substrate, will increase following a permanent cessation of smoking.²⁴

Genetic Polymorphism

The response to xenobiotics and to coadministration of inhibitory or inducing xenobiotics is highly variable. The translation of DNA sequences into proteins results in the phenotypic expression of the genes. When a genetic mutation occurs, the changed DNA may continue to exist, be eliminated, or propagate into a polymorphism. A polymorphism is a genetic change that exists in at least 1% of the human population.^28,59 A polymorphism in a biotransformation enzyme may change its rate of activity. The heterogeneity of CYP enzymes contributes to the differences in metabolic activity between patients.²⁸ Differences in biotransformation capacity that lead to toxicity, once thought to be “idiosyncratic” drug reactions, are likely caused by these inherited differences in the genetic complement of individuals.

The normal catalytic speed of CYP enzyme activity is called extensive. There are two major metabolizer phenotypes due to polymorphism: poor (slow) and ultraextensive (rapid).^28,56 The CYP2C19 and CYP2D6 genes are highly polymorphic (Table 13–1). The CYP2D6 gene, which has more than 90 alleles, is associated with both ultraextensive and poor metabolism. The CYP2C19 and CYP2C9 genes are both associated with polymorphisms resulting in poor metabolizers.^10,59

The clinical implications of polymorphisms are vast. A prodrug may not be bioactivated because the patient is a poor metabolizer. Conversely, a drug may not reach a therapeutic concentration because the patient is an ultraextensive metabolizer.²⁸

Polymorphisms exist for enzymes other than CYP enzymes. A classic one is the inheritance of rapid or slow “acetylator” phenotypes. Acetylation is important for the biotransformation of amines (R–NH₂) or hydrazines (NH₂–NH₂). Slow acetylators are at increased risk of toxicity associated with the slower biotransformation of certain nitrogen-containing xenobiotics such as isoniazid, procainamide, hydralazine, and sulfonamides.⁷⁸

Polymorphic genes that code for enzymes in important metabolic pathways affect the toxicity of a xenobiotic by altering the response to, or the disposition of, the xenobiotic. An example occurs in glucose-6-phosphate dehydrogenase (G6PD) deficiency. G6PD is a critical enzyme in the hexose monophosphate shunt, a metabolic pathway located in the red blood cell (RBC) that produces NADPH, which is required to maintain RBC glutathione in a reduced state. In turn, reduced glutathione prevents hemolysis during oxidative stress.¹² In patients deficient in G6PD, oxidative stress produced by electrophilic xenobiotics results in hemolysis.

Induction of CYP Enzymes

Biotransformation by induced CYP enzymes results in either increased activity of prodrugs or enhanced elimination of drugs. Stopping an inducing agent may result in the opposite effects. Either way, maintaining therapeutic concentrations of affected drugs is difficult, resulting in either toxicity or subtherapeutic concentrations. Interestingly, not all CYP enzymes are inducible. The inducible enzymes include CYP2A, CYP2B, CYP2C, CYP2E, and CYP3A.⁵⁰

While varied mechanisms of induction exist, the most common and significant is nuclear receptor (NR)-mediated increase in gene transcription.⁵⁰ NRs are the largest group of transcription factors (proteins) that switch genes on or off.⁸⁷ They regulate reproduction, growth, and biotransformation enzymes, including CYP enzymes.⁸⁴ NRs exist mostly within the cytoplasm of cells. The CYP families 2 and 3 both have gene activation triggered through the NR pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). The CYP 1A subfamily uses the aryl hydrocarbon receptor (AhR) as its NR. Ligands, molecules that bind to and affect the reactivity of a central molecule, are typically small and lipophilic, enabling them to enter cells. Many xenobiotics are ligands. Ligands bind the NRs, resulting in structural changes that enable the NR-ligand complexes to be translocated into the cell nucleus. Within the nucleus, NR-ligand complexes bind to proteins such as the retinoid X receptor (RXR), shared by the PXR and the CAR, or the AhR nuclear translocator (Arnt), or by the AhR. This new complex then interacts with specific response elements of DNA, initiating the transcription of a segment of DNA, and resulting in the phenotypic expression of the respective CYP enzyme.

The ligand-binding domain of the PXR is very hydrophobic and flexible, enabling this pocket to bind many substrates of varied sizes and reflecting why the PXR can be activated by a broad group of ligands.^65,84 For example, xenobiotic ligands that bind the NR PXR that targets the CYP3A4 gene include rifampin, omeprazole, carbamazepine, and troleandomycin. Phenobarbital, a classic inducing xenobiotic, is a ligand that binds the CAR.⁸⁷ The induction of CYP1A subfamily enzymes is through the interaction with the NR AhR. Exogenous AhR ligands are hydrophobic, cyclic, planar molecules. Classic AhR ligands include polycyclic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene.⁶⁵

Induction requires time to occur because it involves de novo synthesis of new proteins. Similarly, withdrawal of the inducer results in a slow return to the original enzyme concentration.⁵⁰ Polyaromatic hydrocarbons (PAHs) result in CYP1A subfamily induction within 3 to 6 hours with a maximum effect within 24 hours.⁸⁰ The inducer rifampin does not affect verapamil trough concentrations maximally until one week; followed by a two-week return to baseline steady state after withdrawal of rifampin.⁵⁰ Xenobiotics with long half-lives require longer periods to reach steady-state concentrations that maximize induction. Phenobarbital and fluoxetine, which have long half-lives, may fully manifest induction only after weeks of exposure. Conversely, xenobiotics with short half-lives, such as rifampin or venlafaxine, can reach maximum induction within days.¹⁰

Inconsistency in CYP induction exists in all organs and among individuals.^50,65 In an in vitro study of the effects of inducing xenobiotics on 60 livers, differences in enzyme induction ranged from 5-fold for CYP3A4 and CYP2C9 up to more than 50-fold for CYP2A6 and CYP2D6.⁵⁰ The inconsistency likely results from both polymorphisms and multiple environmental factors including diet, tobacco, and pollutants.⁵⁰ There is variation in the extent to which inducers can generate new CYP enzymes. Identical dosing regimens with rifampin have resulted in induction of in vivo hepatic CYP3A4 with up to 18-fold differences between subjects.⁵⁰ There is an inverse correlation of the degree of inducibility of an enzyme and the baseline enzyme concentration. Patients with a relatively low baseline concentration of a CYP enzyme will be more inducible than those with a high baseline concentration. Interestingly, the maximum concentrations of CYP enzymes seem to be quantitatively similar among individuals, suggesting a limit to which enzymes can be induced.⁵⁰

Although the focus of this section is on CYP enzymes, it appears that all phases of xenobiotic metabolism are regulated by NRs.⁸ Also, just as genetic polymorphisms exist for CYP enzymes, they exist for NRs including the AhR, the CAR, and the PXR. This results in varied sensitivities to the ligands that complex with the NRs, ultimately resulting in differences in CYP enzyme induction.^50,87

Inhibition of CYP Enzymes

CYP enzyme inhibition can result in increased bioavailability of a drug or decreased bioactivation of a prodrug.⁶⁶ Inhibition of CYP enzymes is the most common cause of harmful drug–drug interactions.⁶⁶ Inhibition of CYP enzymes by coadministered xenobiotics has resulted in the removal of many medications from the market in recent years, including terfenadine, mibefradil, bromfenac, astemizole, cisapride, cerivastatin, and nefazodone.⁹² The appendix at the end of this chapter includes a comprehensive listing of cytochrome P450 substrates, inhibitors, and inducers.

Inhibition mechanisms include irreversible (mechanism-based inhibition) and the more common reversible processes. The most common type of reversible inhibition is competitive, where the substrate and inhibitor both bind the active site of the enzyme.^66,92 Binding is weak and is formed and broken down easily, resulting in the enzyme becoming available again. It occurs rapidly, usually beginning within hours.¹⁰ Because the degree of inhibition varies with the concentration of the inhibitor, the time to reach the maximal effect correlates with the half-life of the xenobiotic in question.¹⁰ A competitive inhibitor can be overcome by increasing the substrate concentration. Each substrate of a CYP enzyme is an inhibitor of the metabolism of all the other substrates of the same enzyme, thereby increasing their concentrations and half-lives. Reversible, noncompetitive inhibition occurs when an inhibitor binds a location on an enzyme that is not the active site, resulting in a structural change that inhibits the active enzyme site. For example, noncompetitive inhibitors of CYP2C9 include nifedipine, tranylcypromine, and medroxyprogesterone.⁷⁷ Another reversible mechanism results from competition between one xenobiotic and a metabolite of a second xenobiotic at its CYP enzyme substrate binding site. For example, the metabolites of clarithromycin and erythromycin produced by CYP3A inhibit further CYP3A activity. The effect is reversible and usually increases with repeated dosing.⁷⁷ Some reversible inhibitors bind so tightly to the enzyme that they essentially function as irreversible inhibitors.⁶⁶

Irreversible inhibitors have reactive groups that covalently, and thus permanently, bind the enzyme. They display time-dependent inhibition because the amount of active enzyme at a given concentration of irreversible inhibitor will be different depending on how long the inhibitor is preincubated with the enzyme. Because the enzyme will never be reactivated, inhibition lasts until new enzyme is synthesized.⁶⁶ One measure of inhibitor potency is the inhibitory concentration, K_i, the concentration of the inhibitor that produces 50% inhibition of the enzyme. The more potent the inhibitor, the lower the value.⁸⁰ Values below 1 µmol/L are regarded as potent.⁶² The azole antifungals are very potent, with K_i values of 0.02 µmol/L.⁸⁰

The impact of an inhibitor is also affected by the fraction of the substrate that is biotransformed by the inhibited, target enzyme. The inhibition of a CYP enzyme will have little impact if the enzyme only metabolizes a fraction of the affected drug.⁶² Conversely, drugs that are primarily metabolized by a single CYP enzyme are more susceptible to interactions.⁶⁶ Simvastatin is mainly biotransformed by CYP3A4. The potent and specific CYP3A inhibitor itraconazole prevents its metabolism, resulting in an increased risk of rhabdomyolysis.¹

Specific CYP Enzymes

CYP1A1 and 1A2.

While 1A1 is located primarily in extrahepatic tissue, 1A2 is a hepatic enzyme and is involved in the metabolism of 10% to 15% of all pharmaceuticals.^10,50 They both are very inducible by polycyclic aromatic hydrocarbons, including those in cigarette smoke and charred food. They bioactivate several procarcinogens including benzo[a]pyrene.⁴³ Xenobiotics activated by the CYP1 enzyme family in the GI tract are linked to colon cancer.⁵⁹

CYP2C9.

The CYP2 enzyme family, with its 76 alleles, is one of the most polymorphic of the CYP enzymes (Table 13–1).^24,88 The CYP2C9 enzyme is the most abundant enzyme of the CYP2C enzyme subfamily, which with CYP2C19, comprises approximately 10% to 20% of the CYP enzymes in the liver and is involved in 15% to 20% of all drug metabolism.^24,88 This enzyme biotransforms S-warfarin, the more active isomer of warfarin. Warfarin is one of the most commonly reported causes of adverse drug events. There is an association between slow metabolism and an increased risk of bleeding in patients taking warfarin.^13,76

CYP2D6.

This enzyme is of historical significance because the exploration of differences in pharmacokinetics among individuals taking the antihypertensive debrisoquine was an important part of the beginning of pharmacogenetics. This enzyme is highly polymorphic, with more than 90 allelic variants.⁶³ Twenty-five percent of pharmaceuticals, including 50% of the commonly used antipsychotics, are substrates for CYP2D6.^38,59,63 Because CYP2D6 is the only drug-metabolizing CYP enzyme that is noninducible, the polymorphisms are the primary reason for the substantial interindividual variation in enzyme activity.⁶³

CYP2E1.

This enzyme comprises 7% of the total CYP enzyme content in the human liver.⁵⁸ It metabolizes small organic compounds, including ethanol, carbon tetrachloride, and halogenated anesthetics.⁸⁰ It also biotransforms low-molecular-weight xenobiotics including benzene, acetone, and N-nitrosamines.⁸⁰ Some of its substrates are procarcinogens, which are bioactivated by CYP2E1. Besides CYP1A2, this is the only other CYP enzyme linked to cancer.⁵⁹ The assessment for a relationship between CYP2E1 and cancer is intense because many of its substrates are environmental xenobiotics. The induction of CYP2E1 is associated with increased liver injury by reactive metabolites of carbon tetrachloride and vinyl chloride (Chap. 23).³⁰ During the metabolism of substrates that include carbon tetrachloride, ethanol, acetaminophen (APAP), aniline, and N-nitrosomethylamine, CYP2E1 actively produces free radicals and other reactive metabolites associated with adduct formation and lipid peroxidation (Chaps. 9 and 35).¹⁴ CYP2E1 is inhibited by acute elevations of ethanol, an effect illustrated by the acute administration of ethanol inhibiting the metabolism of APAP.³⁰ The chronic ingestion of ethanol hastens its own metabolism through CYP2E1 induction.

CYP3A4.

CYP3A4 is the most abundant CYP in the human liver, comprising 40% to 55% of the mass of hepatic CYP enzymes.^10,24 The CYP3A4 enzyme is the most common one found in the intestinal mucosa and is responsible for much first-pass drug metabolism.¹⁰ It is involved in the biotransformation of 50% to 60% of all pharmaceuticals.^58,97 It has broad substrate specificity because it accommodates large lipophilic substrates and can adopt multiple conformations. It can even simultaneously fit two relatively large compounds (ketoconazole, erythromycin) in its active site.⁷⁵

Methadone, extensively metabolized by CYP3A4, is associated with many examples of adverse drug interactions.⁴¹ Torsade de pointes is reported in methadone patients who are also exposed to CYP3A4 inhibitors including ciprofloxacin,⁵⁷ itraconazole,⁶¹ and the antiretrovirals atazanavir and ritonavir.²³ Ketoconazole inhibits CYP3A4, causing a 15-fold to 72-fold increase in serum concentrations of terfenadine.⁵⁹ Bioflavonoids in grapefruit juice decrease metabolism of some substrates by 5-fold to 12-fold.^10,59 The CYP3A4 enzyme does not exhibit significant genetic polymorphism; however, there are large interindividual variations in enzyme concentrations that can affect metabolic rates.⁹⁷

Phase II Biotransformation Reactions

Phase II biotransformation reactions are synthetic, catalyzing the conjugation of products of phase I reactions or xenobiotics with endogenous molecules that are generally hydrophilic. Conjugation usually terminates the pharmacologic activity of xenobiotics and greatly increases their water solubility and excretability.^52,83,95 Conjugation occurs most commonly with glucuronic acid, sulfates, and glutathione. Less common phase II reactions include conjugation with amino acids such as glycine, glutamic acid, and taurine as well as acetylation and methylation.

Glucuronidation is the most common phase II synthesis reaction.⁵² Glucuronyl transferase has relatively low substrate affinity but it has high capacity at higher substrate concentrations.⁸³ The glucuronic acid, donated by uridine diphosphate glucuronic acid, is conjugated with the nitrogen, sulfhydryl, hydroxyl, or carboxyl groups of substrates. Smaller conjugates usually undergo renal elimination, whereas larger ones undergo biliary elimination.⁴⁸

Sulfation complements glucuronidation because it is a high-affinity but low-capacity reaction that occurs primarily in the cytosol. For example, the affinity of sulfate for phenol is very high (the K_m is low), so that when low doses of phenol are administered, the predominant excretion product is the sulfate ester. Because the capacity of this reaction is readily saturated, glucuronidation becomes the main method of detoxification when high doses of phenol are administered.^52,95 Sulfate conjugates are highly ionized and very water soluble. Of note, sulfation is reversible by the action of sulfatases within the liver. The resultant metabolites may be resulfated, and the cycle may repeat itself further.⁹⁵

Glutathione S-transferases are important because they catalyze the conjugation of the tripeptide glutathione (glycine-glutamate-cysteine, or GSH) with a diverse group of reactive, electrophilic metabolites of phase ICYP enzymes. The reactive compounds initiate an attack on the sulfur group of cysteine, resulting in conjugation with GSH that detoxifies the reactive metabolite. Of the three phase II reactions addressed, hepatic concentrations of glutathione by far account for the greatest amount of cofactors used. Although intracellular glutathione is difficult to deplete, when it does occur, severe hepatotoxicity often follows.⁹⁵ Some GSH conjugates are directly excreted. More commonly, the glycine and glutamate residues are cleaved and the remaining cysteine is acetylated to form a mercapturic acid conjugate that is readily excreted in the urine. A familiar example of this detoxification is the avid binding of N-acetyl-p-benzoquinoneimine (NAPQI), the toxic metabolite of APAP, by glutathione.^4,9

As with the CYP enzymes, many phase II enzymes are inducible. For example, UDP-glucuronosyltransferase, which performs glucuronidation, is inducible via the PXR, CAR, and AhR nuclear receptors after binding with rifampin, phenobarbital, and PAHs, respectively. Its activity varies 6-fold to 15-fold in liver microsomes.⁸⁷

Membrane Transporters

Although the focus on drug disposition has traditionally been on biotransformation, membrane transporter proteins greatly impact drug disposition.³⁵ Transporter proteins mediate the cellular uptake and efflux of endogenous compounds and xenobiotics.⁹⁸ Their baseline physiologic role is to transport sugars, lipids, amino acids, and hormones so as to regulate cellular solute and fluid balance. Biotransformation cannot occur unless xenobiotics are taken up into the cells via transport proteins. After xenobiotics have undergone phase I and II metabolism, the metabolites undergo transport protein mediated efflux from the cell, an action that has been called phase III metabolism.³⁹

The transport proteins that are most relevant to xenobiotic uptake and efflux are those mediating the efflux from the apical (luminal) membrane of enterocytes (P-glycoprotein), the uptake from the portal venous blood into the hepatocytes, the efflux from the hepatocytes via the canalicular membrane into the bile, the uptake into renal proximal tubular cells, and the efflux from renal proximal tubular cells into urine.⁹⁸ Drug disposition is facilitated or prevented by the transport proteins.⁴⁴

Most transporters are in the adenosine triphosphate binding cassette (ABC) family of transmembrane proteins that use energy from ATP hydrolysis.^11,35 This family includes the P-glycoprotein family. Some transporters move substrates both into and out of cells. Organs important for drug disposition have multiple transporters that have overlapping substrate capabilities, a redundancy that enhances protection. In the small intestine, P-glycoprotein is important because it can actively extrude xenobiotics back into the intestinal lumen.¹¹ The degree of phenotypic expression of P-glycoprotein affects the bioavailability of many xenobiotics, including paclitaxel, digoxin, and protease inhibitors. Hepatocyte efflux transporters move biotransformed xenobiotics into bile. Transporters in endothelial cells of the blood–brain barrier prevent CNS entry of substrate xenobiotics.^11,35

As with biotransformation enzymes and nuclear receptors, membrane transporters may also be inhibited or induced. Digoxin, a high-affinity substrate for P-glycoprotein, has increased bioavailability when administered with P-glycoprotein inhibitors such as clarithromycin or atorvastatin.³⁵ Loperamide is a substrate for P-glycoprotein that limits its intestinal absorption or CNS entry. Coadministration with quinidine, a P-glycoprotein inhibitor, results in increased opioid CNS effects of loperamide.³⁵ As with the biotransformation enzymes, polymorphisms exist for membrane transporters. However, the clinical significance of these is not clear.¹⁶

Ideally and commonly, potentially toxic metabolites produced by phase I reactions are detoxified during phase II reactions. However, detoxification does not always occur. This section reviews mechanisms of cellular injury related to xenobiotic biotransformation.

Synthesis of Toxins

Sometimes a xenobiotic is mistaken for an endogenous substrate by synthetic enzymes that biotransform it into an injurious compound. The incorporation of the rodenticide fluoroacetate into the tricarboxylic acid cycle is an example of this mechanism of toxic injury (Fig. 13–3).⁶⁹ Another example is illustrated by analogs of purine or pyrimidine bases that are phosphorylated and inserted into growing DNA or RNA chains, resulting in mutations and disruption of cell division. This mechanism is used therapeutically with 5-fluorouracil (5-FU), an antitumor, pyrimidine base analog. When phosphorylated to 5-fluoro-dUTP and incorporated into growing DNA chains, it causes structural instability of the cellular DNA and inhibits tumor growth.⁷³

Injury by Metabolites of Biotransformation

Many toxic products result from metabolic activation (Table 13–2).⁴⁰ The CYP enzymes most associated with bioactivation are 1A1, 1B1, 2A6, and 2E1, whereas 2C9 and 2D6 yield little toxic activation.³²

Highly reactive metabolites exert damage at the site where they are synthesized; reacting too quickly with local molecules to be transported elsewhere. This commonly occurs in the liver, the major site of biotransformation of xenobiotics^31,79 (Chap. 23). However, metabolism in the lungs, skin, kidneys, GI tract, and nasal mucosa can also create toxic metabolites that cause local injury.^9,46 Overdoses of APAP lead to excessive hepatic production of the highly reactive electrophile NAPQI, which initiates a damaging covalent bond with hepatocytes⁴ (Chap. 35). Acute renal tubular necrosis also occurs in patients with overdose of APAP, attributed to its biotransformation by prostaglandin H synthase within renal tubular cells to a highly reactive semiquinoneimine.^22,91

Monoamine oxidases (MAOs) are mitochondrial enzymes present in many tissues. They oxidize many amines, including dopamine, epinephrine, and serotonin, and xenobiotics such as primaquine and haloperidol. The metabolic activity of MAOs was responsible for the outbreak of parkinsonism associated with the use of methylphenyltetrahydropyridine (MPTP), an unintended by-product of attempts to synthesize a “designer” analog of meperidine, methylphenylpropionoxypiperidine (MPPP). After crossing the blood–brain barrier, MPTP is biotransformed by MAO in glial cells to methylphenyldihydropyridine (MPDP⁺), which is converted to MPP⁺. The MPP⁺ is subsequently taken up by specific dopamine transport systems into dopaminergic neurons in the substantia nigra, resulting in inhibition of oxidative phosphorylation and subsequent neuronal death.²⁹

Free Radical Formation

Within an atom, it is energetically favorable for electrons to exist in pairs or as a part of a chemical bond. An element or compound with an unpaired electron, called a radical or free radical, is highly reactive and short lived. It rapidly seeks other species in order to obtain another electron. Radicals include the superoxide anion O₂•^–, which is produced by adding an electron to O₂, and the highly reactive hydroxyl radical HO•, which is produced by splitting the H₂O₂ molecule into two equal halves. The H₂O₂ molecule itself is reactive and is also associated with injury. The superoxide and hydroxyl radicals react with other molecules in order to stabilize themselves; however, by taking an electron in order to do so, they generate new free radical species, potentially initiating a chain reaction. The production of radicals is a normal occurrence for which the human body has defense mechanisms. However, some xenobiotics promote the formation of reactive oxidant species to the extent that antioxidant mechanisms are overwhelmed, a condition called oxidative stress. Oxidizing species are called such because, by reducing themselves by taking away electrons, they oxidize the species from which they took the electrons.⁶

Although oxidative stress may result in oxidative damage to nucleic acids and proteins, other classic targets are polyunsaturated fatty acids (PUFAs) in cellular membranes, resulting in lipid peroxidation (the oxidative destruction of lipids). This attack removes the particularly reactive hydrogen atom, with its lone electron, from a methylene carbon of a PUFA, leaving an unpaired electron and causing the formation of a lipid radical. This lipid radical attacks other PUFAs, initiating a chain reaction that destroys the cellular membrane. Membrane degradation products initiate inflammatory reactions in the cells, resulting in further damage.^6,79

Molecular oxygen (O₂) has a lone pair of electrons in its orbit. Because oxygen is a relatively weak univalent electron acceptor (and most organic molecules are weak univalent electron donors), oxygen cannot efficiently oxidize amino acids and nucleic acids. However, the unpaired electrons of O₂ readily interact with the unpaired electrons of transition metals and organic radicals. Metals frequently catalyze the creation of oxygen free radicals. Figure 13–4 reflects the typical steps in the formation of a hydroxyl radical. The damaging effects of the free radicals are decreased by reaction with antioxidants such as ascorbate, tocopherols, and glutathione.⁵² Deficiencies of antioxidants, especially glutathione, are associated with increased oxidative damage. Free radicals are also neutralized by several enzymes, including peroxidase, superoxide dismutase, and catalase.

The ethanol-inducible CYP2E1 enzyme produces significant amounts of superoxide and peroxide free radicals, and, in the presence of iron, hydroxyl free radicals that readily initiate lipid peroxidation. This has been studied extensively in models of the metabolism of carbon tetrachloride, ethanol, and APAP.¹⁹ The formation of free radicals is implicated in the pulmonary injury caused by paraquat, the myocardial injury caused by doxorubicin, and the liver injury caused by carbon tetrachloride.^55,70 Paraquat reacts with NADPH to form a pyridinyl free radical, which, in turn, reacts with oxygen to generate the superoxide anion radical. Doxorubicin is metabolized to a semiquinone free radical in the cardiac mitochondria, which, in the presence of oxygen, forms a superoxide anion radical that initiates myocardial lipid peroxidation.⁵⁵ Carbon tetrachloride (CCl₄) is metabolized to the trichloromethyl radical (•CCl₃) that binds covalently to cellular macromolecules. In the presence of oxygen, this is converted to the trichloromethylperoxyl radical (•CCl₃O₂) that can initiate lipid peroxidation (Fig. 13–5).⁷¹ See Chap. 108 for a more extensive discussion.

Energy metabolism is the foundation of cellular function. It provides high-energy fuel, predominantly in the form of ATP, for all energy-dependent cellular processes such as synthesis, active transport, and maintenance of electrolyte balance and membrane integrity. Numerous pathways interconnect glycogen, fat, and protein reserves in many tissues that store and retrieve ATP and glucose. The brain and RBCs are entirely dependent on glucose for energy production, while other tissues can also use ketone bodies and fatty acids to synthesize ATP. Rapid cell death occurs if the production or use of ATP is inhibited, thus the goal of many metabolic processes is the production and mobilization of cellular energy.

Catabolic pathways, those that break down molecules into smaller units enabling the production of cellular energy, include glycolysis, the citric acid (tricarboxylic acid, or Krebs) cycle, and oxidative phosphorylation via the electron transport chain. Glycolysis produces small amounts of ATP through the anaerobic metabolism of glucose. Pyruvate, the end product of glycolysis, yields far more ATP when it is converted to acetylcoenzyme A(acetyl-CoA) and “processed” in the citric acid cycle (Fig. 13–3). Fat and protein yield their energy through their conversion to acetyl-CoA and other intermediates of the citric acid cycle. The citric acid cycle and oxidative phosphorylation, via the electron transport chain, result in most ATP synthesis. Oxidative phosphorylation disposes of electrons or “reducing equivalents” and converts their energy to ATP. A lack of oxygen stops the electron transport chain and ATP production. Oxidative metabolism is highly energy efficient, producing 36 moles of ATP for each mole of glucose metabolized, compared to the 2 moles of ATP produced by anaerobic glycolysis. The following sections review the basics of cellular energy metabolism and several important xenobiotics that affect these critical metabolic functions (Table 13–3).^24,45

Glycolysis

Glycolysis is the first biochemical pathway in the metabolism of glucose. Other sugars enter this pathway after conversion to glycolytic intermediates (Fig. 13–6). The glycolytic process converts one molecule of glucose to two of pyruvate plus two molecules of ATP plus two molecules of NADH. Pyruvate may follow many paths. Under anaerobic conditions, the two pyruvate molecules produced from one glucose molecule are reduced by lactate dehydrogenase to two lactate molecules in an NADH-requiring step that regenerates NAD⁺. Thus, anaerobic glycolysis yields two molecules of lactate plus two molecules of ATP. When NAD⁺ and oxygen are available, pyruvate is transported from the cytosol into the mitochondria where it enters the citric acid cycle by being converted by pyruvate decarboxylase to acetyl-CoA (Fig. 13–3).^24,45 In energy-rich conditions, pyruvate is used in fatty acid synthesis, whereas in energy-poor situations, it is used in gluconeogenesis.

Arsenate (As⁵⁺) affects the glycolytic step where 3-phosphoglyceraldehydedehydrogenase (3-PGA) catalyzes the oxidation of glyceraldehyde-3- phosphate to 1,3-diphosphoglycerate, a reaction that preserves a high-energy phosphate bond used to synthesize ATP in the next step of glycolysis (Fig. 13–6).³⁶ Arsenate acts as an analog of phosphate at this step, resulting in an unstable arsenate intermediate that is rapidly hydrolyzed to 3-phosphoglycerate. While glycolysis continues, there is the loss of ATP synthesis because 1,3 biphosphoglycerate is not made.³⁶

Citric Acid Cycle

The citric acid cycle uses acetyl-CoA derived from glycolysis, fat, or protein to regenerate NADH from NAD⁺. The cycle is a major source of electrons (in the form of NADH) and is critical to the aerobic production of ATP (Fig. 13–3). Each acetyl-CoA molecule that is oxidized within the citric acid cycle ultimately forms one molecule each of CO₂ and guanosine triphosphate (GTP), and more importantly, three molecules of NADH and one molecule of reduced flavin adenine dinucleotide (FADH₂), which enter the electron transport chain, producing a total of 12 molecules of ATP. In addition, the citric acid cycle provides important intermediates for amino acid synthesis and for gluconeogenesis.⁴⁵

Various xenobiotics inhibit the citric acid cycle. The rodenticides, sodium fluoroacetate and fluoroacetamide, are combined with coenzyme A, CoASH, to create fluoroacetyl CoA (FAcCoA). The FAcCoA substitutes for acetyl CoA, entering the TCA cycle by condensation with oxaloacetate to form fluorocitrate. Fluorocitrate metabolism results in a metabolite that blocks aconitase within the cycle, resulting in citrate accumulation and the termination of oxidative metabolism (Fig. 13–3) (Chap. 115).

Thiamine is an important cofactor for two citric acid cycle enzymes: the conversion of pyruvate to acetyl-CoA by pyruvate decarboxylase and for the conversion of α-ketoglutarate to succinyl-CoA by α-ketoglutarate dehydrogenase (Fig. 13–3).²⁴ The life-threatening effects of thiamine deficiency are likely related to impairment of these enzyme functions (Antidotes in Depth: A24). Arsenite (As³⁺) inhibits these thiamine-dependent enzymes within the citric acid cycle.⁶⁹

The Electron Transport Chain

The electron transport chain is the location where the “phosphorylation” of oxidative phosphorylation occurs. Oxidative phosphorylation is the creation of high-energy bonds by phosphorylation of ADP to ATP, “coupled” to the transfer of electrons from reduced coenzymes to molecular oxygen via the electron transport chain. The success of aerobic metabolism requires the disposal of electrons within NADH and FADH, generated by oxidative metabolism within glycolysis and the citric acid cycle. The electron transport chain consists of a series of cytochrome–enzyme complexes within the inner mitochondrial membrane (Fig. 13–3). Within these complexes, NADH is split into NAD⁺ + H⁺ + two electrons at complex I at the beginning of the chain while FADH₂ is split into FADH + H⁺ + two electrons at complex II. These reactions have two results. First, the regenerated NAD⁺ and FADH are recycled back to the citric acid cycle, enabling oxidative metabolism to continue. Second, these actions provide the energy required to pump protons (H⁺) from the mitochondrial matrix into the intermembrane space. This action causes the matrix to become relatively alkaline compared to the now acidified intermembrane space and the creation of a proton gradient across the inner mitochondrial membrane. The final step in the electron flow of oxidative phosphorylation is the reduction of molecular oxygen to water by complex IV, cytochrome a-a₃ (Fig. 13–3).^24,45 This hydrogen ion gradient provides the energy needed to create the high-energy bonds of ATP at complex V.

Mitochondria oxidize substrates, consume oxygen, and make ATP. Xenobiotics that interrupt oxidative phosphorylation impair ATP production either by inhibiting specific electron chain complexes or by acting as “uncouplers.” Both of these mechanisms result in rapid depletion of cellular energy stores, followed by failure of ATP-dependent active transport pumps, loss of essential electrolyte gradients, and increases in cell volume.²⁵

Inhibitors of specific cytochromes block electron transport and cause an accumulation of reduced intermediates proximal to the site of inhibition. This stops the regeneration of oxidized substrates for the citric acid cycle, particularly NAD⁺ and FADH, further impairing oxidative metabolism. Cyanide, carbon monoxide, and hydrogen sulfide block the cytochrome a-a₃–mediated reduction of O₂ to H₂O. The very dramatic clinical effects of these exposures illustrate the importance of aerobic metabolism (Chap. 126). Other xenobiotics are less commonly associated with inhibition of the electron transport chain (Table 13–3).^85,89

Severe metabolic acidosis is a clinical manifestation of xenobiotics that inhibit aerobic respiration. This metabolic acidosis is primarily caused by the accumulation of protons in the mitochondrial matrix that are not used in the production of ATP. While lactic acid accumulates, it is only a marker for metabolic acidosis associated with the impairment of oxidative metabolism.⁷⁴

Xenobiotics that uncouple oxidative phosphorylation, like inhibitors of the electron transport chain, reduce ATP synthesis. However, with uncouplers, protons continue to be pumped into the intermembrane space, electrons continue to flow down the chain to reduce oxygen, and substrate consumption continues. Uncouplers dissipate the proton gradient across the mitochondrial inner membrane by allowing the protons to cross back into the mitochondrial matrix. Since it is the proton gradient that drives the production of ATP at complex V, ATP production is reduced. Thus, oxygen consumption is “uncoupled” from ATP production. The redox energy created by electron transport that cannot be coupled to ATP synthesis is released as heat. Various xenobiotics uncouple ATP synthesis (Table 13–3). A classic one is dinitrophenol, used in the past as an herbicide and as a weight-loss product (Chap. 42). Xenobiotics that are capable of carrying hydrogen ions across membranes are generally lipophilic weak acids. These xenobioticsmust have an acid-dissociable group to carry the proton and a bulky lipophilic group to cross a membrane.⁴⁷ Dinitrophenol is able to carry its proton from the cytosol into the more alkaline mitochondrial matrix where it dissociates, acidifying the matrix and destroying the proton gradient across the inner mitochondrial membrane. Interestingly, the phenolate anion of dinitrophenol is relatively lipophilic and can cross back out to the cytosol where it gains a new proton and starts the process over again. Long-chain fatty acids uncouple oxidative phosphorylation by a similar mechanism.^47,89 Fatal exposures to dinitrophenol and to pentachlorophenol, a wood preservative, are associated with severe hyperthermia attributed to heat generation by uncoupled oxidative phosphorylation.⁶⁸ The hyperthermia and acidosis associated with severe salicylate poisoning are attributed to its uncoupling of oxidative phosphorylation.⁸¹

Hexose Monophosphate Shunt

The hexose monophosphate shunt provides the only source of cellular NADPH. NADPH is used in biosynthetic reactions, particularly fatty acid synthesis, and is an important source of reducing power for the maintenance of sulfhydryl groups that protect the cell from free radical injury.^7,37 As noted earlier, G6PD is a key enzyme in the pathway (Fig. 13–7). Reduced glutathione, which is quantitatively the most important antioxidant in cells, depends on the availability of NADPH. RBCs are especially vulnerable to deficiency of NADPH, which results in hemolysis during oxidative stress.

Another manifestation of oxidative stress in RBCs is the oxidation of the iron in hemoglobin from Fe²⁺ to Fe³⁺, producing methemoglobin that occurs both spontaneously and as a response to xenobiotics such as nitrites and aminophenols. Because most reduction of methemoglobin is done by NADH-dependent methemoglobin reductase, which is not deficient in persons who lack G6PD, such persons do not develop methemoglobinemia under normal circumstances. However, when oxidative stress is severe and methemoglobinemia develops, people who have G6PD deficiency have limited ability to use the alternative NADPH-dependent methemoglobin reductase (Chap. 127).⁸⁶

Gluconeogenesis

Gluconeogenesis facilitates the conversion of amino acids and intermediates of the citric acid cycle to glucose. It occurs primarily in the liver but also in the kidney. It is an important source of glucose during fasting and enables maintenance of glycogen stores. Most of the steps in the synthesis of glucose from pyruvate are simply the reverse of glycolysis, with three irreversible exceptions: (1) the conversion of glucose-6-phosphate to glucose; (2) the conversion of fructose-1,6-diphosphate to fructose-6-phosphate; and (3) the synthesis of phosphoenolpyruvate from pyruvate. The synthesis of phosphoenolpyruvate from pyruvate is especially complex. Pyruvate is first converted to oxaloacetate within the mitochondria; then to malate, which is transported out of the mitochondria and converted in the cytosol back to oxaloacetate; and then to phosphoenolpyruvate (Fig. 13–8). Certain amino acids—notably alanine, glutamate, and aspartate—are readily converted to citric acid cycle intermediates and can be used in the synthesis of glucose through this cycle.⁴⁵ Glycerol, produced by the breakdown of triglycerides in adipose tissue, is another substrate for gluconeogenesis.

The regulation of gluconeogenesis is opposite to that of glycolysis, stimulated by glucagon and catecholamines but inhibited by insulin. Gluconeogenesis requires the cytosolic NAD⁺ and mitochondrial NADH. It is impaired by processes that increase the cytosol-reducing potential as measured by the cytosol NADH/NAD⁺ ratio (see discussion below).

A number of xenobiotics impair gluconeogenesis, resulting in hypoglycemia when glycogen stores are depleted (Table 13–3). Hypoglycin A, an unusual amino acid found in unripe ackee fruit that is the cause of Jamaican vomiting sickness, produces profound hypoglycemia.⁸¹ Its metabolite methylenecyclopropylacetic acid (MCPA) indirectly inhibits gluconeogenesis by blocking the oxidation of long-chain fatty acids, an important source of NADH in mitochondria. It also inhibits the metabolism of several glycogenic amino acids, including leucine, isoleucine, and tryptophan, and blocks their entrance into the citric acid cycle. MCPA may also prevent the transport of malate out of the mitochondria.^72,81,82 Hypoglycemia also occurs in fasting patients with elevated ethanol concentrations.^3,27,46 This is likely a result of the impairment of gluconeogenesis by the increased cytosolic NADH:NAD⁺ ratio associated with the metabolism of ethanol. This inhibits the two cytosolic steps that require NAD⁺—the conversions of lactate to pyruvate and of malate to oxaloacetate.^2,46

Fatty Acid Metabolism

Fatty acid metabolism occurs primarily in hepatocytes. Fatty acids mobilized in adipose tissue enter hepatocytes by passive diffusion. Fatty acid synthesis is stimulated by insulin and inhibited by glucagon and epinephrine. Acetyl-CoA is the primary building block of free fatty acids (FFAs). In energy-repleted cells, fatty acids are combined with glycerol phosphate to form triacylglycerol (triglycerides), the first step in the synthesis of fat for storage. Hepatic triglycerides are bound to lipoprotein to form very-low-density lipoprotein and then transported and stored in adipocytes. When hepatocytes are energy depleted, triglycerides are broken down to FFAs and glycerol. This process is suppressed by insulin but supported by glucagon or epinephrine. FFAs undergo β-oxidation in the mitochondria, a process that breaks the FFA into acetyl-CoA molecules that can then enter the citric acid cycle. FFAs require activation before transport into the mitochondria. This is accomplished by acylcoenzyme A (acyl-CoA) synthetase, which adds a CoA group to the FFA in an energy-dependent synthetic reaction. These are transported into the mitochondria by a process that utilizes cyclical binding to carnitine, a “carnitine shuttle” (Fig. 13–3). Once inside the mitochondria, FFAs are converted to acetyl-CoA by β-oxidation, involving the sequential removal of two-carbon fragments, each time acting at the second carbon (the β carbon) position of the fatty acid. Each two-carbon molecule removed from the FFA produces one NADH and one FADH₂, which are oxidized in the electron transport chain, and one mole of acetyl-CoA, which enters the citric acid cycle. This process produces 1.3 times more ATP per molecule of carbon metabolized than does the oxidative metabolism of glucose or other carbohydrates.⁴⁵

Many xenobiotics interrupt fatty acid metabolism at various steps, resulting in accumulation of triglycerides in the liver (Table 13–3; Fig. 13–9). The mechanisms of disruption of fatty acid metabolism are poorly defined.¹⁵ Some xenobiotics, including ethanol, hypoglycin, and nucleoside analogs, inhibit β-oxidation, at least indirectly, through effects on NADH concentrations. Protease inhibitors are associated with a syndrome of peripheral fat wasting, central adiposity, hyperlipidemia, and insulin resistance.

The condition of alcoholic ketoacidosis is related in part to inhibition of gluconeogenesis in the alcoholic patient and in part to an exuberant response to nutritional needs by the fatty acid machinery. Vomiting in the alcoholic patient leads to decreased intake of carbohydrate, which stimulates a starvation response with increases in serum glucagon, cortisol, growth hormone, and epinephrine concentrations, and decreases in serum insulin. When the need for carbohydrate is not met by gluconeogenesis, lipolysis, which is normally inhibited by insulin, is intensified and fatty acid mobilization progresses. Glucagon stimulates mitochondrial carnitine acyltransferase, and β-oxidation of fatty acids is increased. The increased mitochondrial NADH:NAD⁺ ratio favors the production of β-hydroxybutyrate over acetoacetate, its oxidized form. The administration of fluids, dextrose, and thiamine to the alcoholic patient leads to correction of this process.⁵³

• Biotransformation is a complex process, involving numerous enzyme systems, that usually detoxifies xenobiotics, however, “metabolic activation” may occur.

• Phase I reactions add functional groups to lipophilic xenobiotics to enhance their reactivity, preparing them for further detoxification by synthetic phase II reactions that enhance hydrophilicity and renal elimination.

• The phase I cytochrome P450 (CYP) enzymes biotransform most xenobiotics; CYP3A4 is the most abundant one, metabolizing 50% to 60% of all pharmaceuticals.

• CYP enzyme activity is quite variable due to genetic polymorphisms, genetic changes that exist in at least 1% of the human population. Enzyme activity also varies because of induction and inhibition.

• The most common method of induction is a nuclear receptor-mediated increase in gene transcription. Inhibition of CYP enzymes is the most common cause of harmful drug–drug interactions, the most common type being competitive, where the substrate and inhibitor both bind the active site of the enzyme.

• Drug disposition is also effected by membrane transporter (MT) proteins that mediate the cellular uptake and efflux of xenobiotics.

• P-glycoprotein is an MT protein that mediates the efflux of xenobiotics from the apical (luminal) membrane of intestinal enterocytes.

References

References