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Chemical factors that influence toxicity
Chapter 4
Chemical factors that influence toxicity The primary determinants of whether or not a compound is toxic are its chemical structure and the resulting physical properties. To maintain homeostasis necessary for life, all biological organisms maintain physical membranous barriers that regulate the translocation of compounds (Chapter 3). These membranous barriers play an important role in ensuring that compounds possessing specific chemical properties can enter or exit the organism, while others cannot. The ability of a chemical to move across or disrupt the normal function of cell membranes is a major determinant of toxicity. The diffusion rate of a chemical across cell membranes is related to its relative solubility in the aqueous and lipid phases of cell membranes. Some chemicals can diffuse across or disrupt cell membranes and interact directly with electrophilic or nucleophilic groups in biomolecules (DNA, RNA, protein, or lipid), leading to their inactivation, degradation, or precipitation. Example compounds include corrosive chemicals, strong acids or bases, oxidizers, chorine, ozone, and disinfectants. Molecules that are highly polar or charged (e.g., ions) and therefore not soluble in lipid cannot cross the cell membrane by simple diffusion. This means that charged ions such as Na+ and Cl must be transported across the membrane via specific transport mechanisms. Some molecules can be actively transported into cells against their concentration gradients via cotransporter systems. For example, active transport of glucose against concentration gradient occurs via Na+/glucose cotransporter that simultaneously move the sugar and Na+ into cells. In this way, the energy for moving sugar against a concentration gradient is provided by the movement of Na+ into cells down its concentration gradient. Levels of ions including Ca++ and Na+ must be maintained at different levels inside and outside the cell by active transporters that require the use of energy. Inhibition of these transporters or preventing the utilization of ATP as an energy source breaks down concentration gradients allowing water to enter the cells, causing swelling and cell death. Compounds that are more soluble in lipid can diffuse across cell membranes rapidly and efficiently. Molecules such as ethyl alcohol and ethers that are less polar can rapidly equilibrate across cell membranes, while their oxidized forms (e.g., carboxylic acids) that are ionized at physiological pH must be actively transported across membranes.
Loomis’s Essentials of Toxicology. https://doi.org/10.1016/B978-0-12-815921-7.00004-1 © 2020 Elsevier Inc. All rights reserved.
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Since all living tissues and cells are capable of carrying on metabolic processes, cells possess the ability to alter (biotransform) many endogenous and exogenous compounds. For example, glucose molecules entering the cells are phosphorylated by specific kinase in the cytoplasm. This is the first step in glycolysis, a process by which sugar molecules are converted into energy and reducing power to synthesize other biological molecules. Phosphorylation also makes the sugar derivative more polar at physiological pH, preventing its diffusion out of cells. Biotransformations are catalyzed by enzymes. While many enzymes are highly specific for their substrates, others are more promiscuous, binding to and catalyzing the biotransformation of many different compounds. Examples of such enzymes are Phase I metabolic enzymes that are expressed in all organisms from bacteria to man. These molecules generally oxidize both endogenous and exogenous chemicals by adding hydroxyl and carboxylic groups, making them more polar. Biotransformation can lead to the activation or detoxification of toxicants or drugs. In higher organisms, the products of Phase I biotransformation can be further conjugated by Phase II enzymes to add large polar groups such as UDP-glucuronate, sulfate, acetates, methyl groups, or the tripeptide glutathione. These modifications make metabolites highly soluble in water and allow their excretion via specific transport mechanisms. In summary, toxicity of chemicals is primarily determined by their structures and physical and chemical properties. These properties are important because they regulate the translocation of chemicals throughout biologic tissues. Chemical structures of compounds or their metabolites also determine their ability to react with biomolecules and to induce toxicity by altering the structures and functions of biomolecules. The ability to predict biological activities of a compound based on its chemical structure is the basis for quantitative structure–activity relationships (QSAR), an important analytical approach widely used in predictive toxicology.
Nonspecific chemical action The mechanisms underlying chemical-induced adverse effects vary from a generalized destruction of protein to specific inhibition of enzyme systems. Strong acids, corrosive agents, and highly reactive oxidizers can produce generalized destruction of living cells by direct chemical reactions. This nonspecific action, sometimes called necrotic cells death, is induced by concentrated solutions of caustic and corrosive chemicals that produce indiscriminate cell destruction. A generalized overwhelming action of this type is no different from those resulting from “burning” the tissue. These chemically induced lesions are commonly referred as “chemical burns.” Such effects are produced not only by strong acids or bases applied in harmful concentrations but also by exposure to concentrated solutions of organic solvents, such as ether, chloroform, or carbon tetrachloride. The intensity of such nonspecific toxicity is directly related to concentrations of
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the chemical agents when they are in contact with the target tissues. A generalized destruction of cells can be produced by any chemical that is sufficiently soluble in tissue fluids to gain access to the cells at high concentrations. Actions of these chemicals in higher organisms are usually limited to readily accessible tissues such as the skin, eyes, mouth, nasal membranes, and pulmonary airways.
Selective chemical action In contrast to the nonspecific cell necrosis induced by highly reactive chemicals, many chemicals of interest in toxicology and pharmacology have more selective modes or mechanisms of action. As a result, the effects of these compounds are more insidious, and predicting their toxicity requires considerable knowledge of chemistry and interactions with biological systems.
Target concepts in toxicity A selective chemical action is when chemicals can interact with normal constituents of cells to change cell activities or functions. These normal constituents of cells are referred to as “targets” for the assaulting chemicals. A chemical may target a specific reactive group on many biomolecules. An example of such a mode of action is the chemical reaction of mercury cations with the sulfur group on cysteine residues in a protein. Mercury reacts with a specific amino acid side chain, but this reaction can occur in any protein containing this residue. Another example is planar molecules (e.g., mutagen ICR141) that nonspecifically intercalates between base pairs of double-stranded DNA to alter its structure and function. Selective inactivation or denaturation of a specific class of biomacromolecules leads to loss of critical cellular functions. Some chemicals can interact with a single protein or a highly related class of proteins with high specificity and affinity. These interactions typically occur by binding the compound to protein at a specific conformational structure or binding pocket, leading to inactivation of functions associated with the macromolecules. These interactions can, for example, target specific structures in nucleic acid sequences. For example, antisense oligonucleotides (e.g., gapmers), or siRNA molecules can target specific RNA or DNA sequences, resulting in inactivation of RNA or DNA. Medicines (e.g., ganciclovir) and other nucleoside analogs can interact with DNA polymerase and become incorporated into nascent nucleic acid chains. But because of their chemical structures, the modified nascent chains are not substrates for further elongation, leading to chain termination and replicative arrest. Toxicants such as lectins can bind to specific side chains on glycosylated proteins and impair maturation or function of the proteins. While specific targets for toxicants can be part of any essential macromolecule or macromolecular complex, most toxicants target proteins. A variety of natural products, pesticides, and drugs can target specific proteins essential to
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signal transduction in neuronal cells. Among the earliest studied was the poison curare extracted from various plants by natives in Central and South America. This alkaloid was used to generate poison arrows used in hunting. Curare activates acetylcholine signaling pathway by binding to nicotinic acetylcholine receptors found in the neuromuscular junctions and subsequently causes muscular weakness, paralysis, and even death by asphyxiation due to paralysis of the diaphragm. Other toxicants can target the same neuronal signaling pathway by different mechanisms. For example, organophosphate pesticide chlorpyrifos activates acetylcholine signaling pathway by inhibiting acetylcholine esterase, which normally degrades acetylcholine in the synapses to minimize the signal. In other cases, neurotransmitters (e.g., dopamine, GABA, and serotonin) are actively taken up into presynaptic nerve endings by specific transporters. A classic example of a chemical targeting reuptake of the neurotransmitter dopamine from the synapse and inducing neuronal cell death is 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MTPT). This chemical induced rapid onset of Parkinsonian disease when it was inadvertently injected as a contaminant by illicit use of the drug, heroin. Targeting specific proteins is also widely exploited in developing antiinfectious agents. For example, ganciclovir specifically targets the DNA polymerase of herpes virus while having little affinity for the mammalian DNA polymerase. Another example is penicillin, the first antibiotic discovered. Penicillin kills gram-positive bacteria (Bacillus anthraces, Clostridium, Listeria, and Streptococcus) by specifically inhibiting the transpeptidase enzyme involved in the synthesis of the cell wall necessary for bacterial growth. The cell wall comprises proteoglycan polymers, and transpeptidase cross-links these polymers to form a thick mesh of fibers resembling cellulose. In the presence of penicillin, the cell wall weakens, allowing water to access the cell membrane, leading to bacterial death. Gram-negative bacteria (e.g., Escherichia coli, Yersinia pestis, and Neisseria gonorrhoeae) have much thinner and denser cell walls that do not require the same transpeptidase system for its synthesis. Thus, penicillin is used to selectively treat patients infected with gram-positive bacteria that have the transpeptidase system.
Effect of ionization and lipid solubility on translocation of chemicals Many drugs and toxicants are weak organic acids or bases and hence are ionized at physiological pH. As already discussed, ions and many large molecules enter cells by interacting with specific transporters in the cell membrane. Other compounds in their nonionized forms can pass cell membranes by diffusion across the lipid bilayer. The degree of ionization of a compound in aqueous solution is dependent upon the pH. The pH of an aqueous solution can be adjusted so that the compound exists half in the ionized form and half in the nonionized form. That pH is the acidic dissociation constant (or pKa) of the compound. At a pH above the pKa of a compound, acids exist in aqueous solution mainly in the
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ionic form, and bases exist in the nonionic form. Conversely, at a pH below the pKa of a compound, acids exist in aqueous solution mainly in the nonionic form, and bases are in the ionic form. The pKa of a compound may be derived from the Henderson–Hasselbalch equation as follows: for acids pKa ¼ pH + log
nonionized form ionized form
for bases pKa ¼ pH + log
ionized form nonionized form
If the pKa of an acid or base is known and the pH of the aqueous solution of the compound is known, it is possible to calculate the ratio of the ionized to nonionized forms of the chemical in solution. If two aqueous solutions of an electrolyte at different concentrations are separated by a biologic membrane that is only permeable to the nonionized molecules, in time, a state of equilibrium will be established across the membrane. At equilibrium, the concentrations of the ionized and nonionized forms of the compound in each solution are identical but only if the pH values of the two solutions are identical. The concentrations of the compound on different sides of the membrane will be different if the pH values of the solutions are different. The concentrations of the electrolyte on the two sides of the membrane can be determined by the two pH values. The pH on both sides of a cellular membrane in most biologic specimens is essentially the same. If a nonionized compound that can pass through the membrane is introduced to one side of the membrane, its movement across the membrane depends on the pH. A compound that is highly ionized at ambient pH would fail to traverse the membrane. Compounds that exist primarily in the nonionized state at physiological pH would be expected to diffuse through membranes, provided that the nonionized form is lipid soluble. Diffusion will occur down the existing concentration gradient until equilibrium is reached across the membrane. When a difference in pH exists on opposing sides of a membrane, a concentration gradient will be created with regard to the nonionized moiety. At equilibrium, the total quantity of electrolyte may be many times greater on one side of the membrane than on the other side. In the warm-blooded mammal, there are two sites at which the pH on the opposing sides of membranes may differ greatly. These include the mucosal surface of the gastrointestinal tract and the lumen of the tubules of the kidney. At these sites, the effect of pH on the ionization of organic electrolytes controls their transfer of the electrolytes and ionized compounds across the membrane. In this way, pH controls absorption of charged compounds across the gastrointestinal tract and their excretion of compound by the kidney. Fig. 4.1 illustrates the proportion of nonionized to ionized forms of acetylsalicylic acid (pKa 3.5) in the stomach (pH 1.0), in the intestine (pH 5.3), in the interstitial fluid or blood (pH 7.4), in acid urine (pH 6.0), and in alkaline urine (pH 7.0) at equilibrium for the different pH in each
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FIG. 4.1 Proportions of nonionized and ionized forms of acetylsalicylic acid (pKa ¼ 3.5) in biologic fluids.
compartment. This simplified example (Fig. 4.1) does not, however, take into account the effect of facilitated or active transport. When a chemical is introduced into the stomach or the intestine, a concentration gradient for the chemical exists between the site of deposition and other compartments. Absorption progresses in the direction of the concentration gradient. If only the nonionized form is absorbed across the compartment barriers, the amount of the nonionized form determines the rate of absorption of the compound. The low pH of the stomach would highly favor transport of ionized form of drugs that are weak acids across the stomach mucosal membrane to the blood. Although the absorption of aspirin from the stomach is complex, one of the factors is stomach pH. Experimental results confirm that the rate of absorption of aspirin (acetylsalicylic acid) and its metabolite (salicylic acid) into human body fluids is efficient in stomach and intestine, where the pHs are acidic. The higher pHs seen in the kidney and urine favor excretion. Hence, under physiological conditions, the kidney would be expected to be a poor organ for excretion of this drug. If alkaline urine is produced in the kidney, the concentration gradient for the nonionized form favors excretion of the drug from the blood to the urine. By contrast, acidification of urine is formed in the kidney; the concentration gradient of the nonionized acetylsalicylic acid favors transfer of the drug from the urine back into the blood. The effect of urinary pH on the excretion of acetylsalicylic acid from a dog is shown in Fig. 4.2. Excretion of acetylsalicylic acid more than quadrupled as the pH of the urine increased from 6.7 to 7.8. In pharmacology, it is common practice to refer to the gaseous and vapor anesthetic agents (such as ether, chloroform, cyclopropane, nitrous oxide, ethylene, and divinyl ether) as being “nonreactive” with tissue constituents. These agents are absorbed, translocated, and excreted from the body with little or no change in their chemical structure. Except for minor losses of the compounds
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FIG. 4.2 Effect of urinary pH on excretion of acetylsalicylic acid (ASA) by the kidney. Dog, male, 12 kg, hydrated with water (250 ml orally) 1 h before the experiment. ASA (0.5 g orally) given 45 min before the experiment. ASA (50 mg) in 0.9% NaCl in water (150 ml) continuously infused during experiment. At 20–25-min NaHCO3 (0.2 g) given intravenously. At 65-min NaHCO3 (0.5 g) given intravenously. Urine collections are made at 15-min intervals from an inlying urinary bladder catheter, and each urine sample includes 50-ml water used to wash the bladder.
through the kidney and sweat, these agents are recovered primarily from the expired air of experimental animals. The mechanisms by which these agents produce anesthesia are not exactly known, but in general, anesthetic potency is directly related to lipid solubility. One theory is that these agents accumulate in the lipid of nerve cells, inhibiting their ability to respond to stimuli. Agents that are inhaled through the pulmonary system diffuse across the pulmonary membrane according to Fick’s law, which states that a gas will diffuse in the direction of a decreasing partial pressure gradient at a rate that is directly proportional to the diffusion coefficient and inversely proportional to the square root of the molecular weight or density of the compound. Thus, the rate of diffusion of such agents into the blood of mammals is proportional to the partial pressure of the agent in the inhaled air. Absorption of the drug from the air to the blood is highly efficient as “the blood makes one circuit through the lungs.” The concentration of anesthetic agents in each tissue is directly proportional to blood supply, as well as water and fat content of each tissue (which determines the solubility coefficients for the various tissues). Nervous tissues have no special affinity for these anesthetic agents other than their increased lipid content. Thus, as long as an anesthetic agent is administered to the animal, a shift in distribution of the agent among animal tissues will continue until equilibrium is established throughout the animal. When administration is discontinued, the body eliminates the agent because the diffusion pressure gradients are reversed. The rate of elimination of these agents is proportional to the partial pressures of the agents in the tissues, blood, and inhaled air. By contrast, inhaled compounds that undergo ionization in biologic fluids would be expected to be
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absorbed, translocated, and excreted by the organism according to the conditions described for electrolytes. In contrast to clinical use of anesthesia, exposures to various gases and vapors may occur over extended periods of time due to chronic air pollution or exposure to contaminated workplace air (e.g., inhalation of solvents by nail salon workers or inhalation of toxic metal vapors by welders). The same principles that determine the absorption and translocation of the gaseous and vapor anesthetic agents would also apply in these situations. However, chronic exposures may result in toxicities that are not observed with transient exposures. Recognizing this potential for harm, the American Industrial Hygiene Association (see Chapter 15) has published estimates for maximal allowable safe concentrations over a normal 8-h daily period for more than 500 chemical agents that may be inhaled from the air at the workplace. A discussion of the basis for these estimates is given in Chapter 5.
Effects of biotransformation on mechanisms of toxicity Biotransformation is the process that converts the parent molecules to which the organism is exposed into more hydrophilic compounds readily excretable in the urine or bile. Biotransformation is catalyzed by enzymes that often show significant substrate promiscuity, allowing for metabolism of both endogenous and exogenous (xenobiotics) chemicals. For example, the esterase-hydrolyzing enzyme, cholinesterase, not only hydrolyzes acetylcholine (a normally occurring neurohormone) but also hydrolyzes the anesthetic agent, procaine, and the muscle-paralyzing drug, succinylcholine. Another example is the monoamine oxidase (MAO) that is not only important in the metabolism of normally occurring biologic amines such as epinephrine and tyramine but also oxidizes foreign short-chain amines such as benzylamine. Most organisms including bacteria express enzyme systems that induce the biotransformation of chemicals. The genes encoding these enzymes are known to be shared by horizontal transmission in bacteria. In most organisms, metabolic enzymes are present as multiple isoforms that exhibit a large amount of genetic diversity. These enzymes can be highly promiscuous with respect to substrate specificity, often metabolizing classes of related or structurally similar compounds. Genetic evidence suggests that this diversity is the result of evolutionary processes (e.g., gene duplication and positive selection for variants) that allow organisms to survive from exposure to a multitude of environmental toxicants. Toxicologists first became interested in xenobiotic metabolizing enzymes because the enzymes play a major role in drug inactivation. These enzymes were initially classified as “drug-metabolizing enzymes” or “drug detoxication enzymes,” but these terms are misleading. These enzymes catalyze biotransformation of many endogenous and exogenous compounds that are not drugs. Moreover, the reactions they catalyze do not always result in detoxication and in many cases are responsible for generating products that are more toxic
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than parent compounds. The terms of “biotransformation enzymes” and “metabolic enzymes” are now preferred in toxicology. Biotransformation enzymes are distributed throughout the body, and most of them are harbored within the smooth endoplasmic reticulum (microsomes) of cells and other subcellular compartments, such as mitochondria and cytosol. The highest concentrations of most biotransformation enzymes are found in the liver, which receives most xenobiotics absorbed from the gastrointestinal tract via the portal circulation. The liver also serves as a major site for metabolism of both endogenous and exogenous chemicals that find their way into the circulatory system. Not all tissues express all biotransformation enzymes suggesting tissue-specific roles in the metabolism of substrates. Biotransformations are catalyzed by Phase I and Phase II enzymes. Phase I enzymes catalyze oxidation, reduction, and hydrolysis. These reactions usually lead to the addition of polar functional group(s) (-OH, -SH, -NH2 or -COOH) that make chemicals more polar and slightly water soluble. The major Phase I enzyme system comprises cytochrome P450 enzymes. They include a large family of proteins named “cyto” for cell plus “chrome” because of their spectroscopic properties due to inclusion of a heme- or iron-containing prosthetic in the enzyme. Cytochrome P450s metabolize a wide range of chemicals through oxidative mechanisms. These heme-containing enzymes generate reactive oxygen species that react with specific electrophilic center in substrates. Cytochrome P450s are present in most tissues, and the highest concentration is found in the liver microsomes. In recent years, much has been learned about the function of these enzymes and their actions on xenobiotics through the discovery of selective inhibitors of cytochrome P450s. Phase II enzymes catalyze glucuronidation, acetylation, sulfation, methylation, conjugation with glutathione, and conjugation with amino acids. Most Phase II biotransformation reactions lead to a large increase in hydrophilicity of compounds, allowing for excretion in urine or bile. Fig. 4.3 illustrates representative metabolic transformation mechanisms found across a variety of species. Phase II biotransformation reactions may or may not be preceded by Phase I biotransformation. These enzymes are not expressed in lower organisms. Biotransformation of chemicals often involves multiple enzymatic steps, the sequence of which may determine toxicity. For example, acetaminophen is a widely used analgesic and antipyretic agent which is considered safe at therapeutic doses. However, overdose can cause hepatic centrilobular necrosis in both humans and experimental animals. As shown in Fig. 4.4, acetaminophen is first metabolized by cytochrome P450 enzymes to its reactive metabolite, N-acetybenzoquinoneimine, which is responsible for acetaminophen-induced centrilobular necrosis of the liver. At normal acetaminophen dosing, Nacetybenzoquinoneimine immediately binds to intracellular glutathione, and the glutathione derivative is eliminated in the urine (detoxification). But with increased dose of acetaminophen, enhanced production of N-acetybenzoquinoneimine
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FIG. 4.3—CONT’D
further depletes glutathione. Once glutathione is depleted, the toxic metabolite, N-acetybenzoquinoneimine, then covalently binds to proteins in the liver and induces hepatocellular toxicity. Acetaminophen can also be metabolized via a minor pathway that involves its sulfation by sulfotransferases or glucuronidation by UDP-glucuronosyltransferases.
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FIG. 4.3 Some types and examples of biotransformation mechanisms in animals. The oxidation and reduction reactions are catalyzed by liver microsomal enzyme systems. The hydrolysis, acetylation, and conjugation reactions may involve enzyme systems from other tissues.
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FIG. 4.4 Acetaminophen metabolism and probable mechanism of hepatic toxicity.
Another good example is aflatoxin, one of the most mutagenic and carcinogenic chemicals that is produced by certain strains of fungi (Aspergillus) growing on grains. The aflatoxin B1 molecule is metabolized primarily by cytochrome p450 enzymes to generate mutagenic aflatoxin B1 8,9-epoxide. Studies suggest that the extent of glutathione conjugation of aflatoxin B1 8,9-epoxide is a major factor in influencing the risk of different animal species to aflatoxin B1-induced hepatocellular carcinoma. In species such as the mouse that express high levels of glutathione-S-transferase, the epoxide is rapidly inactivated by glutathione conjugation and secreted in the urine. In primates and rats, lower levels of glutathione conjugation allow the reactive epoxide to persist and bind to DNA to form DNA adducts, leading to increased risk of carcinogenesis. The take-home message is that both endogenous and exogenous compounds can be metabolized by different biotransformation enzymes and not all of which lead to the formation of toxic metabolites. The relative abundance or activity of these enzymes in different cell types or species can determine which metabolic pathways are preferentially formed and consequently contributing to differential organ and species sensitivities to toxicity.
Inhibition of biotransformation enzymes Various environmental or endogenous conditions can influence the level of biotransformation enzymes and thereby alter toxicity of a specific compound.
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For example, low-protein diet can suppress cytochrome P450 levels and associated enzyme activity. A classic example is the effect of fasting on glutathione levels. Mice are relatively resistant to the hepatotoxic effects of acetaminophen due to high levels of glutathione. However, if mice are fasted for a 12-h period prior to exposure, the levels of glutathione in the liver are depleted, preventing conjugation and inactivation of the reactive intermediate of the drug. As a result, the toxicity of acetaminophen increases dramatically in mice after fasting. There is also ample evidence that the livers of newborn and immature animals are deficient in the microsomal enzymes, making them more susceptible to toxicants. Inhibition of biotransformation enzymes can significantly impact chemicalinduced efficacy and toxicities. One of the first examples of a nonselective inhibitor of cytochrome P450s was Proadifen (SKF-525A). This compound increases the duration of barbiturate-induced anesthesia, probably by directly inactivating the biotransformation enzymes responsible for terminating the action of barbiturates. The effect of SKF-5254 is not limited to altered barbiturate metabolism. Rather, it is a generalized inhibitor of many xenobiotics via independent mechanisms. For example, pretreatment of mice with SKF-525A increases procaine toxicity and decreases the LD50 of procaine from 188 to 79 mg/kg, probably via its effects on transmembrane calcium flux. When biotransformation forms more toxic metabolite than that of the parent compound, inhibition of the biotransformation enzymes would be expected to decrease the toxicity. For example, when animals are pretreated with inhibitors of biotransformation enzymes prior to treatment of acetaminophen, the drug-induced liver necrosis is prevented. Inhibition of biotransformation enzymes plays an important role in drug interactions and can increase toxicity. For example, allopurinol is a xanthine oxidase inhibitor that reduces the synthesis of uric acid in animals and human. Allopurinol is used in the treatment of gout and other clinical conditions associated with increased levels of uric acid. Drugs such as 6-mercaptopurine (an antileukemic drug) and azathioprine (an immunosuppressant drug) are normally inactivated by xanthine oxidase. Concomitant administration of allopurinol can increase the plasma levels of 6-mercaptopurine or azathioprine, leading to profound suppressive effect on hematopoiesis and even death. Another good example is the antihistamine terfenadine that is metabolized by cytochrome P450 (CYP3A4). When terfenadine is administered with ketoconazole or erythromycin (CYP3A4 inhibitors), the plasma level of terfenadine is significantly elevated to block cardiac potassium channel, leading to arrhythmias. Therefore, whenever a drug is a known enzyme inhibitor, toxicity tests should be conducted in combination with other drugs that are dependent on the same enzyme system for their inactivation. Increasing evidences show that compounds in food, natural products, and nutrition supplements (most of which are unregulated) can have significant drug interactions. For example, grapefruit juice can increase toxicity or reduce efficacy of several drugs. Mechanistic studies show that chemicals (e.g.,
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furanocoumarins and flavonoids) in grapefruit juice alter the activity of numerous metabolic enzymes including cytochrome P450s, esterases, and sulfotransferases, as well as organic acid transporter proteins (OATPs) in cell membranes. Clinical data indicate that significant grapefruit juice–drug interactions are mainly mediated by inhibition of CYP3A and OATPs. A few drugs showing strong inhibition of metabolism by grapefruit juice include dihydropyridines (calcium channel antagonists), terfenadine (antihistamine), midazolam and triazolam (benzodiazepine hypnotics), cyclosporine (immunosuppressant), and saquinavir (anti-ADIS drug). Studies provide a precautionary example of how even dietary constituents can affect drug disposition and toxicity. Similarly, precautions should be given to natural products and unregulated nutrition supplements. Patients who take medicines are advised to consult their physicians or pharmacists about drug interactions before taking nutritional supplements or herbal medicines.
Induction of biotransformation enzymes The total quantity of biotransformation enzymes can be increased in humans and in higher animals by prior administration of chemical inducers, such as anesthetics (nitrous oxide, ether, and chloroform), sedatives (barbiturates and urethane), analgesics (glutethimide and phenylbutazone), and hypoglycemic agents or insecticides (chlordane, DDT, dieldrin, aldrin, hexachlorocyclohexane, and heptachlor). Enzyme induction usually requires repeated exposure of the inducing agents, and the effect is usually temporary, lasting from 2 to 4 weeks following the administration of the inducing chemical. Induction of cytochrome P450s can increase the rate of biotransformation and lower the plasma exposure of therapeutic drugs, sometimes leading to ineffective treatment of therapeutic medicines. However, enzyme induction is usually less important than inhibition of cytochrome P450s, because some of the aforementioned examples indicate that enzyme inhibition can cause rapidly elevated drug level in the blood, leading to exaggerated toxicity. Nonetheless, induction of biotransformation enzymes can affect metabolism and toxicity of a variety of compounds. For example, CYP2E1 is one of the principal cytochrome P450s responsible for metabolizing acetaminophen. Alcohol is a known CYP2E1 inducer. Chronic alcohol users may have increased levels of the CYP2E1 enzyme that may metabolize more acetaminophen to the toxic metabolite, N-acetybenzoquinoneimine. The excessive amount of N-acetybenzoquinoneimine might deplete glutathione detoxification pathway, leading to liver damage. The foregoing discussion on the complexity of biotransformation suggests at least two important mechanisms by which chemically induced toxicity or drug efficacy can be altered. First, the toxicity of a given compound can be distinctly different within members of a species or between species. This suggests that toxicity test should always be performed in more than one species. Second,
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inhibition or induction of biotransformation enzymes plays a significant role in altering drug-induced toxicities and drug–drug interaction. Besides being substrates for metabolic enzymes, many medicines can also induce or inhibit metabolic enzymes. Precautions must be taken to control for coexposures that might alter efficacy or toxicity of a given compound.
Chemical factors that influence toxicity The primary determinants of whether or not a compound is toxic are its chemical structure and the resulting physical properties. To maintain homeostasis necessary for life, all biological organisms maintain physical membranous barriers that regulate the translocation of compounds (Chapter 3). These membranous barriers play an important role in ensuring that compounds possessing specific chemical properties can enter or exit the organism, while others cannot. The ability of a chemical to move across or disrupt the normal function of cell membranes is a major determinant of toxicity. The diffusion rate of a chemical across cell membranes is related to its relative solubility in the aqueous and lipid phases of cell membranes. Some chemicals can diffuse across or disrupt cell membranes and interact directly with electrophilic or nucleophilic groups in biomolecules (DNA, RNA, protein, or lipid), leading to their inactivation, degradation, or precipitation. Example compounds include corrosive chemicals, strong acids or bases, oxidizers, chorine, ozone, and disinfectants. Molecules that are highly polar or charged (e.g., ions) and therefore not soluble in lipid cannot cross the cell membrane by simple diffusion. This means that charged ions such as Na+ and Cl must be transported across the membrane via specific transport mechanisms. Some molecules can be actively transported into cells against their concentration gradients via cotransporter systems. For example, active transport of glucose against concentration gradient occurs via Na+/glucose cotransporter that simultaneously move the sugar and Na+ into cells. In this way, the energy for moving sugar against a concentration gradient is provided by the movement of Na+ into cells down its concentration gradient. Levels of ions including Ca++ and Na+ must be maintained at different levels inside and outside the cell by active transporters that require the use of energy. Inhibition of these transporters or preventing the utilization of ATP as an energy source breaks down concentration gradients allowing water to enter the cells, causing swelling and cell death. Compounds that are more soluble in lipid can diffuse across cell membranes rapidly and efficiently. Molecules such as ethyl alcohol and ethers that are less polar can rapidly equilibrate across cell membranes, while their oxidized forms (e.g., carboxylic acids) that are ionized at physiological pH must be actively transported across membranes.
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Since all living tissues and cells are capable of carrying on metabolic processes, cells possess the ability to alter (biotransform) many endogenous and exogenous compounds. For example, glucose molecules entering the cells are phosphorylated by specific kinase in the cytoplasm. This is the first step in glycolysis, a process by which sugar molecules are converted into energy and reducing power to synthesize other biological molecules. Phosphorylation also makes the sugar derivative more polar at physiological pH, preventing its diffusion out of cells. Biotransformations are catalyzed by enzymes. While many enzymes are highly specific for their substrates, others are more promiscuous, binding to and catalyzing the biotransformation of many different compounds. Examples of such enzymes are Phase I metabolic enzymes that are expressed in all organisms from bacteria to man. These molecules generally oxidize both endogenous and exogenous chemicals by adding hydroxyl and carboxylic groups, making them more polar. Biotransformation can lead to the activation or detoxification of toxicants or drugs. In higher organisms, the products of Phase I biotransformation can be further conjugated by Phase II enzymes to add large polar groups such as UDP-glucuronate, sulfate, acetates, methyl groups, or the tripeptide glutathione. These modifications make metabolites highly soluble in water and allow their excretion via specific transport mechanisms. In summary, toxicity of chemicals is primarily determined by their structures and physical and chemical properties. These properties are important because they regulate the translocation of chemicals throughout biologic tissues. Chemical structures of compounds or their metabolites also determine their ability to react with biomolecules and to induce toxicity by altering the structures and functions of biomolecules. The ability to predict biological activities of a compound based on its chemical structure is the basis for quantitative structure–activity relationships (QSAR), an important analytical approach widely used in predictive toxicology.
Nonspecific chemical action The mechanisms underlying chemical-induced adverse effects vary from a generalized destruction of protein to specific inhibition of enzyme systems. Strong acids, corrosive agents, and highly reactive oxidizers can produce generalized destruction of living cells by direct chemical reactions. This nonspecific action, sometimes called necrotic cells death, is induced by concentrated solutions of caustic and corrosive chemicals that produce indiscriminate cell destruction. A generalized overwhelming action of this type is no different from those resulting from “burning” the tissue. These chemically induced lesions are commonly referred as “chemical burns.” Such effects are produced not only by strong acids or bases applied in harmful concentrations but also by exposure to concentrated solutions of organic solvents, such as ether, chloroform, or carbon tetrachloride. The intensity of such nonspecific toxicity is directly related to concentrations of
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the chemical agents when they are in contact with the target tissues. A generalized destruction of cells can be produced by any chemical that is sufficiently soluble in tissue fluids to gain access to the cells at high concentrations. Actions of these chemicals in higher organisms are usually limited to readily accessible tissues such as the skin, eyes, mouth, nasal membranes, and pulmonary airways.
Selective chemical action In contrast to the nonspecific cell necrosis induced by highly reactive chemicals, many chemicals of interest in toxicology and pharmacology have more selective modes or mechanisms of action. As a result, the effects of these compounds are more insidious, and predicting their toxicity requires considerable knowledge of chemistry and interactions with biological systems.
Target concepts in toxicity A selective chemical action is when chemicals can interact with normal constituents of cells to change cell activities or functions. These normal constituents of cells are referred to as “targets” for the assaulting chemicals. A chemical may target a specific reactive group on many biomolecules. An example of such a mode of action is the chemical reaction of mercury cations with the sulfur group on cysteine residues in a protein. Mercury reacts with a specific amino acid side chain, but this reaction can occur in any protein containing this residue. Another example is planar molecules (e.g., mutagen ICR141) that nonspecifically intercalates between base pairs of double-stranded DNA to alter its structure and function. Selective inactivation or denaturation of a specific class of biomacromolecules leads to loss of critical cellular functions. Some chemicals can interact with a single protein or a highly related class of proteins with high specificity and affinity. These interactions typically occur by binding the compound to protein at a specific conformational structure or binding pocket, leading to inactivation of functions associated with the macromolecules. These interactions can, for example, target specific structures in nucleic acid sequences. For example, antisense oligonucleotides (e.g., gapmers), or siRNA molecules can target specific RNA or DNA sequences, resulting in inactivation of RNA or DNA. Medicines (e.g., ganciclovir) and other nucleoside analogs can interact with DNA polymerase and become incorporated into nascent nucleic acid chains. But because of their chemical structures, the modified nascent chains are not substrates for further elongation, leading to chain termination and replicative arrest. Toxicants such as lectins can bind to specific side chains on glycosylated proteins and impair maturation or function of the proteins. While specific targets for toxicants can be part of any essential macromolecule or macromolecular complex, most toxicants target proteins. A variety of natural products, pesticides, and drugs can target specific proteins essential to
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signal transduction in neuronal cells. Among the earliest studied was the poison curare extracted from various plants by natives in Central and South America. This alkaloid was used to generate poison arrows used in hunting. Curare activates acetylcholine signaling pathway by binding to nicotinic acetylcholine receptors found in the neuromuscular junctions and subsequently causes muscular weakness, paralysis, and even death by asphyxiation due to paralysis of the diaphragm. Other toxicants can target the same neuronal signaling pathway by different mechanisms. For example, organophosphate pesticide chlorpyrifos activates acetylcholine signaling pathway by inhibiting acetylcholine esterase, which normally degrades acetylcholine in the synapses to minimize the signal. In other cases, neurotransmitters (e.g., dopamine, GABA, and serotonin) are actively taken up into presynaptic nerve endings by specific transporters. A classic example of a chemical targeting reuptake of the neurotransmitter dopamine from the synapse and inducing neuronal cell death is 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MTPT). This chemical induced rapid onset of Parkinsonian disease when it was inadvertently injected as a contaminant by illicit use of the drug, heroin. Targeting specific proteins is also widely exploited in developing antiinfectious agents. For example, ganciclovir specifically targets the DNA polymerase of herpes virus while having little affinity for the mammalian DNA polymerase. Another example is penicillin, the first antibiotic discovered. Penicillin kills gram-positive bacteria (Bacillus anthraces, Clostridium, Listeria, and Streptococcus) by specifically inhibiting the transpeptidase enzyme involved in the synthesis of the cell wall necessary for bacterial growth. The cell wall comprises proteoglycan polymers, and transpeptidase cross-links these polymers to form a thick mesh of fibers resembling cellulose. In the presence of penicillin, the cell wall weakens, allowing water to access the cell membrane, leading to bacterial death. Gram-negative bacteria (e.g., Escherichia coli, Yersinia pestis, and Neisseria gonorrhoeae) have much thinner and denser cell walls that do not require the same transpeptidase system for its synthesis. Thus, penicillin is used to selectively treat patients infected with gram-positive bacteria that have the transpeptidase system.
Effect of ionization and lipid solubility on translocation of chemicals Many drugs and toxicants are weak organic acids or bases and hence are ionized at physiological pH. As already discussed, ions and many large molecules enter cells by interacting with specific transporters in the cell membrane. Other compounds in their nonionized forms can pass cell membranes by diffusion across the lipid bilayer. The degree of ionization of a compound in aqueous solution is dependent upon the pH. The pH of an aqueous solution can be adjusted so that the compound exists half in the ionized form and half in the nonionized form. That pH is the acidic dissociation constant (or pKa) of the compound. At a pH above the pKa of a compound, acids exist in aqueous solution mainly in the
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ionic form, and bases exist in the nonionic form. Conversely, at a pH below the pKa of a compound, acids exist in aqueous solution mainly in the nonionic form, and bases are in the ionic form. The pKa of a compound may be derived from the Henderson–Hasselbalch equation as follows: for acids pKa ¼ pH + log
nonionized form ionized form
for bases pKa ¼ pH + log
ionized form nonionized form
If the pKa of an acid or base is known and the pH of the aqueous solution of the compound is known, it is possible to calculate the ratio of the ionized to nonionized forms of the chemical in solution. If two aqueous solutions of an electrolyte at different concentrations are separated by a biologic membrane that is only permeable to the nonionized molecules, in time, a state of equilibrium will be established across the membrane. At equilibrium, the concentrations of the ionized and nonionized forms of the compound in each solution are identical but only if the pH values of the two solutions are identical. The concentrations of the compound on different sides of the membrane will be different if the pH values of the solutions are different. The concentrations of the electrolyte on the two sides of the membrane can be determined by the two pH values. The pH on both sides of a cellular membrane in most biologic specimens is essentially the same. If a nonionized compound that can pass through the membrane is introduced to one side of the membrane, its movement across the membrane depends on the pH. A compound that is highly ionized at ambient pH would fail to traverse the membrane. Compounds that exist primarily in the nonionized state at physiological pH would be expected to diffuse through membranes, provided that the nonionized form is lipid soluble. Diffusion will occur down the existing concentration gradient until equilibrium is reached across the membrane. When a difference in pH exists on opposing sides of a membrane, a concentration gradient will be created with regard to the nonionized moiety. At equilibrium, the total quantity of electrolyte may be many times greater on one side of the membrane than on the other side. In the warm-blooded mammal, there are two sites at which the pH on the opposing sides of membranes may differ greatly. These include the mucosal surface of the gastrointestinal tract and the lumen of the tubules of the kidney. At these sites, the effect of pH on the ionization of organic electrolytes controls their transfer of the electrolytes and ionized compounds across the membrane. In this way, pH controls absorption of charged compounds across the gastrointestinal tract and their excretion of compound by the kidney. Fig. 4.1 illustrates the proportion of nonionized to ionized forms of acetylsalicylic acid (pKa 3.5) in the stomach (pH 1.0), in the intestine (pH 5.3), in the interstitial fluid or blood (pH 7.4), in acid urine (pH 6.0), and in alkaline urine (pH 7.0) at equilibrium for the different pH in each
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FIG. 4.1 Proportions of nonionized and ionized forms of acetylsalicylic acid (pKa ¼ 3.5) in biologic fluids.
compartment. This simplified example (Fig. 4.1) does not, however, take into account the effect of facilitated or active transport. When a chemical is introduced into the stomach or the intestine, a concentration gradient for the chemical exists between the site of deposition and other compartments. Absorption progresses in the direction of the concentration gradient. If only the nonionized form is absorbed across the compartment barriers, the amount of the nonionized form determines the rate of absorption of the compound. The low pH of the stomach would highly favor transport of ionized form of drugs that are weak acids across the stomach mucosal membrane to the blood. Although the absorption of aspirin from the stomach is complex, one of the factors is stomach pH. Experimental results confirm that the rate of absorption of aspirin (acetylsalicylic acid) and its metabolite (salicylic acid) into human body fluids is efficient in stomach and intestine, where the pHs are acidic. The higher pHs seen in the kidney and urine favor excretion. Hence, under physiological conditions, the kidney would be expected to be a poor organ for excretion of this drug. If alkaline urine is produced in the kidney, the concentration gradient for the nonionized form favors excretion of the drug from the blood to the urine. By contrast, acidification of urine is formed in the kidney; the concentration gradient of the nonionized acetylsalicylic acid favors transfer of the drug from the urine back into the blood. The effect of urinary pH on the excretion of acetylsalicylic acid from a dog is shown in Fig. 4.2. Excretion of acetylsalicylic acid more than quadrupled as the pH of the urine increased from 6.7 to 7.8. In pharmacology, it is common practice to refer to the gaseous and vapor anesthetic agents (such as ether, chloroform, cyclopropane, nitrous oxide, ethylene, and divinyl ether) as being “nonreactive” with tissue constituents. These agents are absorbed, translocated, and excreted from the body with little or no change in their chemical structure. Except for minor losses of the compounds
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FIG. 4.2 Effect of urinary pH on excretion of acetylsalicylic acid (ASA) by the kidney. Dog, male, 12 kg, hydrated with water (250 ml orally) 1 h before the experiment. ASA (0.5 g orally) given 45 min before the experiment. ASA (50 mg) in 0.9% NaCl in water (150 ml) continuously infused during experiment. At 20–25-min NaHCO3 (0.2 g) given intravenously. At 65-min NaHCO3 (0.5 g) given intravenously. Urine collections are made at 15-min intervals from an inlying urinary bladder catheter, and each urine sample includes 50-ml water used to wash the bladder.
through the kidney and sweat, these agents are recovered primarily from the expired air of experimental animals. The mechanisms by which these agents produce anesthesia are not exactly known, but in general, anesthetic potency is directly related to lipid solubility. One theory is that these agents accumulate in the lipid of nerve cells, inhibiting their ability to respond to stimuli. Agents that are inhaled through the pulmonary system diffuse across the pulmonary membrane according to Fick’s law, which states that a gas will diffuse in the direction of a decreasing partial pressure gradient at a rate that is directly proportional to the diffusion coefficient and inversely proportional to the square root of the molecular weight or density of the compound. Thus, the rate of diffusion of such agents into the blood of mammals is proportional to the partial pressure of the agent in the inhaled air. Absorption of the drug from the air to the blood is highly efficient as “the blood makes one circuit through the lungs.” The concentration of anesthetic agents in each tissue is directly proportional to blood supply, as well as water and fat content of each tissue (which determines the solubility coefficients for the various tissues). Nervous tissues have no special affinity for these anesthetic agents other than their increased lipid content. Thus, as long as an anesthetic agent is administered to the animal, a shift in distribution of the agent among animal tissues will continue until equilibrium is established throughout the animal. When administration is discontinued, the body eliminates the agent because the diffusion pressure gradients are reversed. The rate of elimination of these agents is proportional to the partial pressures of the agents in the tissues, blood, and inhaled air. By contrast, inhaled compounds that undergo ionization in biologic fluids would be expected to be
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absorbed, translocated, and excreted by the organism according to the conditions described for electrolytes. In contrast to clinical use of anesthesia, exposures to various gases and vapors may occur over extended periods of time due to chronic air pollution or exposure to contaminated workplace air (e.g., inhalation of solvents by nail salon workers or inhalation of toxic metal vapors by welders). The same principles that determine the absorption and translocation of the gaseous and vapor anesthetic agents would also apply in these situations. However, chronic exposures may result in toxicities that are not observed with transient exposures. Recognizing this potential for harm, the American Industrial Hygiene Association (see Chapter 15) has published estimates for maximal allowable safe concentrations over a normal 8-h daily period for more than 500 chemical agents that may be inhaled from the air at the workplace. A discussion of the basis for these estimates is given in Chapter 5.
Effects of biotransformation on mechanisms of toxicity Biotransformation is the process that converts the parent molecules to which the organism is exposed into more hydrophilic compounds readily excretable in the urine or bile. Biotransformation is catalyzed by enzymes that often show significant substrate promiscuity, allowing for metabolism of both endogenous and exogenous (xenobiotics) chemicals. For example, the esterase-hydrolyzing enzyme, cholinesterase, not only hydrolyzes acetylcholine (a normally occurring neurohormone) but also hydrolyzes the anesthetic agent, procaine, and the muscle-paralyzing drug, succinylcholine. Another example is the monoamine oxidase (MAO) that is not only important in the metabolism of normally occurring biologic amines such as epinephrine and tyramine but also oxidizes foreign short-chain amines such as benzylamine. Most organisms including bacteria express enzyme systems that induce the biotransformation of chemicals. The genes encoding these enzymes are known to be shared by horizontal transmission in bacteria. In most organisms, metabolic enzymes are present as multiple isoforms that exhibit a large amount of genetic diversity. These enzymes can be highly promiscuous with respect to substrate specificity, often metabolizing classes of related or structurally similar compounds. Genetic evidence suggests that this diversity is the result of evolutionary processes (e.g., gene duplication and positive selection for variants) that allow organisms to survive from exposure to a multitude of environmental toxicants. Toxicologists first became interested in xenobiotic metabolizing enzymes because the enzymes play a major role in drug inactivation. These enzymes were initially classified as “drug-metabolizing enzymes” or “drug detoxication enzymes,” but these terms are misleading. These enzymes catalyze biotransformation of many endogenous and exogenous compounds that are not drugs. Moreover, the reactions they catalyze do not always result in detoxication and in many cases are responsible for generating products that are more toxic
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than parent compounds. The terms of “biotransformation enzymes” and “metabolic enzymes” are now preferred in toxicology. Biotransformation enzymes are distributed throughout the body, and most of them are harbored within the smooth endoplasmic reticulum (microsomes) of cells and other subcellular compartments, such as mitochondria and cytosol. The highest concentrations of most biotransformation enzymes are found in the liver, which receives most xenobiotics absorbed from the gastrointestinal tract via the portal circulation. The liver also serves as a major site for metabolism of both endogenous and exogenous chemicals that find their way into the circulatory system. Not all tissues express all biotransformation enzymes suggesting tissue-specific roles in the metabolism of substrates. Biotransformations are catalyzed by Phase I and Phase II enzymes. Phase I enzymes catalyze oxidation, reduction, and hydrolysis. These reactions usually lead to the addition of polar functional group(s) (-OH, -SH, -NH2 or -COOH) that make chemicals more polar and slightly water soluble. The major Phase I enzyme system comprises cytochrome P450 enzymes. They include a large family of proteins named “cyto” for cell plus “chrome” because of their spectroscopic properties due to inclusion of a heme- or iron-containing prosthetic in the enzyme. Cytochrome P450s metabolize a wide range of chemicals through oxidative mechanisms. These heme-containing enzymes generate reactive oxygen species that react with specific electrophilic center in substrates. Cytochrome P450s are present in most tissues, and the highest concentration is found in the liver microsomes. In recent years, much has been learned about the function of these enzymes and their actions on xenobiotics through the discovery of selective inhibitors of cytochrome P450s. Phase II enzymes catalyze glucuronidation, acetylation, sulfation, methylation, conjugation with glutathione, and conjugation with amino acids. Most Phase II biotransformation reactions lead to a large increase in hydrophilicity of compounds, allowing for excretion in urine or bile. Fig. 4.3 illustrates representative metabolic transformation mechanisms found across a variety of species. Phase II biotransformation reactions may or may not be preceded by Phase I biotransformation. These enzymes are not expressed in lower organisms. Biotransformation of chemicals often involves multiple enzymatic steps, the sequence of which may determine toxicity. For example, acetaminophen is a widely used analgesic and antipyretic agent which is considered safe at therapeutic doses. However, overdose can cause hepatic centrilobular necrosis in both humans and experimental animals. As shown in Fig. 4.4, acetaminophen is first metabolized by cytochrome P450 enzymes to its reactive metabolite, N-acetybenzoquinoneimine, which is responsible for acetaminophen-induced centrilobular necrosis of the liver. At normal acetaminophen dosing, Nacetybenzoquinoneimine immediately binds to intracellular glutathione, and the glutathione derivative is eliminated in the urine (detoxification). But with increased dose of acetaminophen, enhanced production of N-acetybenzoquinoneimine
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FIG. 4.3—CONT’D
further depletes glutathione. Once glutathione is depleted, the toxic metabolite, N-acetybenzoquinoneimine, then covalently binds to proteins in the liver and induces hepatocellular toxicity. Acetaminophen can also be metabolized via a minor pathway that involves its sulfation by sulfotransferases or glucuronidation by UDP-glucuronosyltransferases.
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FIG. 4.3 Some types and examples of biotransformation mechanisms in animals. The oxidation and reduction reactions are catalyzed by liver microsomal enzyme systems. The hydrolysis, acetylation, and conjugation reactions may involve enzyme systems from other tissues.
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FIG. 4.4 Acetaminophen metabolism and probable mechanism of hepatic toxicity.
Another good example is aflatoxin, one of the most mutagenic and carcinogenic chemicals that is produced by certain strains of fungi (Aspergillus) growing on grains. The aflatoxin B1 molecule is metabolized primarily by cytochrome p450 enzymes to generate mutagenic aflatoxin B1 8,9-epoxide. Studies suggest that the extent of glutathione conjugation of aflatoxin B1 8,9-epoxide is a major factor in influencing the risk of different animal species to aflatoxin B1-induced hepatocellular carcinoma. In species such as the mouse that express high levels of glutathione-S-transferase, the epoxide is rapidly inactivated by glutathione conjugation and secreted in the urine. In primates and rats, lower levels of glutathione conjugation allow the reactive epoxide to persist and bind to DNA to form DNA adducts, leading to increased risk of carcinogenesis. The take-home message is that both endogenous and exogenous compounds can be metabolized by different biotransformation enzymes and not all of which lead to the formation of toxic metabolites. The relative abundance or activity of these enzymes in different cell types or species can determine which metabolic pathways are preferentially formed and consequently contributing to differential organ and species sensitivities to toxicity.
Inhibition of biotransformation enzymes Various environmental or endogenous conditions can influence the level of biotransformation enzymes and thereby alter toxicity of a specific compound.
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For example, low-protein diet can suppress cytochrome P450 levels and associated enzyme activity. A classic example is the effect of fasting on glutathione levels. Mice are relatively resistant to the hepatotoxic effects of acetaminophen due to high levels of glutathione. However, if mice are fasted for a 12-h period prior to exposure, the levels of glutathione in the liver are depleted, preventing conjugation and inactivation of the reactive intermediate of the drug. As a result, the toxicity of acetaminophen increases dramatically in mice after fasting. There is also ample evidence that the livers of newborn and immature animals are deficient in the microsomal enzymes, making them more susceptible to toxicants. Inhibition of biotransformation enzymes can significantly impact chemicalinduced efficacy and toxicities. One of the first examples of a nonselective inhibitor of cytochrome P450s was Proadifen (SKF-525A). This compound increases the duration of barbiturate-induced anesthesia, probably by directly inactivating the biotransformation enzymes responsible for terminating the action of barbiturates. The effect of SKF-5254 is not limited to altered barbiturate metabolism. Rather, it is a generalized inhibitor of many xenobiotics via independent mechanisms. For example, pretreatment of mice with SKF-525A increases procaine toxicity and decreases the LD50 of procaine from 188 to 79 mg/kg, probably via its effects on transmembrane calcium flux. When biotransformation forms more toxic metabolite than that of the parent compound, inhibition of the biotransformation enzymes would be expected to decrease the toxicity. For example, when animals are pretreated with inhibitors of biotransformation enzymes prior to treatment of acetaminophen, the drug-induced liver necrosis is prevented. Inhibition of biotransformation enzymes plays an important role in drug interactions and can increase toxicity. For example, allopurinol is a xanthine oxidase inhibitor that reduces the synthesis of uric acid in animals and human. Allopurinol is used in the treatment of gout and other clinical conditions associated with increased levels of uric acid. Drugs such as 6-mercaptopurine (an antileukemic drug) and azathioprine (an immunosuppressant drug) are normally inactivated by xanthine oxidase. Concomitant administration of allopurinol can increase the plasma levels of 6-mercaptopurine or azathioprine, leading to profound suppressive effect on hematopoiesis and even death. Another good example is the antihistamine terfenadine that is metabolized by cytochrome P450 (CYP3A4). When terfenadine is administered with ketoconazole or erythromycin (CYP3A4 inhibitors), the plasma level of terfenadine is significantly elevated to block cardiac potassium channel, leading to arrhythmias. Therefore, whenever a drug is a known enzyme inhibitor, toxicity tests should be conducted in combination with other drugs that are dependent on the same enzyme system for their inactivation. Increasing evidences show that compounds in food, natural products, and nutrition supplements (most of which are unregulated) can have significant drug interactions. For example, grapefruit juice can increase toxicity or reduce efficacy of several drugs. Mechanistic studies show that chemicals (e.g.,
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furanocoumarins and flavonoids) in grapefruit juice alter the activity of numerous metabolic enzymes including cytochrome P450s, esterases, and sulfotransferases, as well as organic acid transporter proteins (OATPs) in cell membranes. Clinical data indicate that significant grapefruit juice–drug interactions are mainly mediated by inhibition of CYP3A and OATPs. A few drugs showing strong inhibition of metabolism by grapefruit juice include dihydropyridines (calcium channel antagonists), terfenadine (antihistamine), midazolam and triazolam (benzodiazepine hypnotics), cyclosporine (immunosuppressant), and saquinavir (anti-ADIS drug). Studies provide a precautionary example of how even dietary constituents can affect drug disposition and toxicity. Similarly, precautions should be given to natural products and unregulated nutrition supplements. Patients who take medicines are advised to consult their physicians or pharmacists about drug interactions before taking nutritional supplements or herbal medicines.
Induction of biotransformation enzymes The total quantity of biotransformation enzymes can be increased in humans and in higher animals by prior administration of chemical inducers, such as anesthetics (nitrous oxide, ether, and chloroform), sedatives (barbiturates and urethane), analgesics (glutethimide and phenylbutazone), and hypoglycemic agents or insecticides (chlordane, DDT, dieldrin, aldrin, hexachlorocyclohexane, and heptachlor). Enzyme induction usually requires repeated exposure of the inducing agents, and the effect is usually temporary, lasting from 2 to 4 weeks following the administration of the inducing chemical. Induction of cytochrome P450s can increase the rate of biotransformation and lower the plasma exposure of therapeutic drugs, sometimes leading to ineffective treatment of therapeutic medicines. However, enzyme induction is usually less important than inhibition of cytochrome P450s, because some of the aforementioned examples indicate that enzyme inhibition can cause rapidly elevated drug level in the blood, leading to exaggerated toxicity. Nonetheless, induction of biotransformation enzymes can affect metabolism and toxicity of a variety of compounds. For example, CYP2E1 is one of the principal cytochrome P450s responsible for metabolizing acetaminophen. Alcohol is a known CYP2E1 inducer. Chronic alcohol users may have increased levels of the CYP2E1 enzyme that may metabolize more acetaminophen to the toxic metabolite, N-acetybenzoquinoneimine. The excessive amount of N-acetybenzoquinoneimine might deplete glutathione detoxification pathway, leading to liver damage. The foregoing discussion on the complexity of biotransformation suggests at least two important mechanisms by which chemically induced toxicity or drug efficacy can be altered. First, the toxicity of a given compound can be distinctly different within members of a species or between species. This suggests that toxicity test should always be performed in more than one species. Second,
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inhibition or induction of biotransformation enzymes plays a significant role in altering drug-induced toxicities and drug–drug interaction. Besides being substrates for metabolic enzymes, many medicines can also induce or inhibit metabolic enzymes. Precautions must be taken to control for coexposures that might alter efficacy or toxicity of a given compound.