The Metabolism of Xenobiotic Chemicals John W. Cullen Monroe Community College, Rochester, NY 14623
Every day humans are exposed to thousands of different chemicals. Of these most are encountered in minute amounts. Many are introduced in the diet, including not onlv nutrients such as proteins, carbohydrates, and fats, hut also a variety of nonnutsients such as food additives used to preserve and flavor foods, pesticides and herbicides used in agriculture, drugs added to animal feed, and a vast array of natural products from plants and microorganisms. Those who live in hiehlv - .industrialized areas are also exoosed to pollutants in air and drinking water. Regardless of their source these exogenous chemicals are termed xenobiotics (from the Greek, meaning "foreign to life"). Since manv xenobiotics are toxic, the body must have a successful strategy to detoxify or eliminate these chemicals from the body if i t is to survive. Once a chemical enters the hlood stream, it is usually eliminated via the kidneys by excretion into the urine. The physiology of the kidney is such, however, that only polar, water-soluble substances are readily eliminated. Nonpolar, lipophilic substances diffuse from the kidnev back into the hlood ( I ) . These substances mlwt I P mrt?hhlizrd to more polar c o m p u ~ ~ n I,cit,re ds heing excrrtt.d. 'Chv mrtr~bolismo i xenohit~tics~ c c u r sinsinhf in the liver by enzymes which are different from the enzymes in most biochemical pathwavs in that they are relatively nonspecific. This is because these enzymes must he versatile enouah to handle substrates with a wide range of functional groups, molecular weights and shapes, lipophilicities, etc. The prohlem is that this detoxicatiun system is not perfect. ~ E c a s i o n a ~metabolite ~~ will he formed that is more toxic than the original xenohiotic. The introduction of polar (often oxygen) functionality into lipophilic molecules often requires metabolic activation to highly reactive intermediates. If these intermediates are not deactivated safely in a controlled, enzymatic process 100% of the time, the organism will he a t enhanced risk. Of particular concern is the generation of epoxides, carhonium ions, nitrenium ions, and other extremely electrophilic species (2) in the region of endogenous cellular nucleophilic molecules such as proteins and nucleic acids. Reaction with these electrophiles can cause destruction of vital cell proteins and mutations in the DNA structure. These events can lead to liver necrosis, cancer, or ot,her serious diseases. When chemical carcinogenesis began to be studied intensively in the 1940's, there was the hope among workers in the field that there would be some common structural feature among chemicals that were carcinogenic. As the numher and variety of known carcinogens increased, it became evident that there would he no simple correlation between some physical or chemical molecular parameter and carcinogenic potency. A major advance in our current understanding of chemical carcinogenesis was made by James and Elizabeth Miller ( 3 ) ,who proposed that most organic carcinogens were molecules that could be transformed into reactive electrophiles by cellular enzymes. The "precarcinogens" require metaholic activation to be converted into the "ultimate carcinogens", which are then thought to hind to nucleophilic sites in DNA. The 1n6,urtnnce of meraholkm was emphasized hv Hrurr Arne< ( 4 1 in the developnwnr of thr Salmonrlln micrwome mutagenicity test (A& test). The test is done by combining a tester strain of histidine-requiring Salmonella mutants, a 396
Journal of Chemical Education
homogenized liver extract from a mammal such as a rat and the putative mutagenic chemical, and incubating a t 37 "C. Mutaeenicitv is determined hv the numher of bacterial colanies that have undergone a reverse mutation to histidineindependent strains. If the liver extract containing the activating enzymes is omitted, many mutagens are not detected. When the test procedures are optimized, the Ames test is estimated to hemore than 90% accurate in detecting chemical mutagens. Most of our knowledge about the reactions involved in the metaholism of xenobiotics is based on research done in drug metaholism. The general strategy of xenohiotic metaholism is the same in all mammalian species, hut there are often strikiue differences in chemical oathwavs from one soecies to another. These differences have important physiological conseauences. In addition there are differences in individuals that affect the metabolism of chemicals. These include grnrtic fnct~rs.age, wx, hormonal state, changes in intestlnnl micmflora, and d~seases(especially liver disease). Williams ( 5 ) in his rlassir text di\,ided the pathways of drug metaholism into two major categories. Phase I reactions (biotransformations) include oxidations, reductions, and hydrolyses. In phase I reactions functional groups are added to the molecule or existing functional groups are modified so as to increase the polarity of the molecule. Depending on the particular xenohiotic, oxygen functionality may he introduced or nitrogen and sulfur functionality unmasked. In phase I1 reactions (conjugations) highly polar, often ionic, arouos .. . such as alucuronic acid, sulfate, acetvl, nnd mrrcapturic acid are addcd to existing oxygen, nitrogen. or sulfur funrtinnaiitv to ~ r o d u r ereadil\, excrrtahlr roniugates. Most xenobiot& undergo both phase I and phase I1 reactions. The must common type of phase I reactions are oxidations. The mixed-function oxidases, or mono-oxygenases, are a family of NADPH-dependent enzymes concentrated in the smooth endoplasmic reticulum of liver cells. These enzvmes catalvze a numher of hvdroxvlation. epoxidation. and other oxidation reactions using 0 2 as the oxidizing agent. An iron atom in the hemoorotein cvtochrome P-450 binds molecular oxygen and activates it for reaction with the organic suhstrate. The exact nature of this iron-oxvpen species is not completely understood, hut it is extremely;eaciive and can insert into C-H, N--H, S-H, and C=C bonds (6). The cytochrome P-450 system is said to be inducible; that is, enzyme levels and metabolic activity increase in response to certain environmental factors. Agents known to induce these enzymes include a number of polycyclic aromatic hydrocarbons found in ciearette smoke. drues such as he nobarbital, many pesticidks including and several comoonents of normal foods such as flavones, indoles, and xanthines. Some of the reactions catalvzed hv cvtochrome P-450 are shown in Figure 1. With alkanes or aikil side chains hydroxvlation is usuallv seen a t the terminal or penultimate carbon atom. For exakple, the major metabolite of n-heptane is 2heptanol (Fig. 1,eq 1).With functionalized substrates oxidation usually occurs adjacent to the functional group. 1-Pentene undergoes hydroxylation in the allylic position and also epoxidation of the alkene (eq 2). Anisole undergoes oxidation a t the methyl group to give a hemiacetal, which is w
converted spontnnrously to phenol and formaldehyde (eq 3). reiulring in an overall 0 dealkylarion. Similarly N-methylaniline is hydroxylntcd to give a carhinolamine, which decomposes tocivr andine and formaldehyde ( e 4), ~ an overall N-dealkylati& reaction. Aromatic hydrocarbons are also oxidized in cytochrome P-450 mediated reactions. Benzene and many substituted benzenes are oxidized to phenols. At first i t was thought that the reaction occurred simply by insertion of an oxygen atom into a carhon-hydrogen bond, hut i t was subsequently shown that arene oxides were intermediates in the reaction (eq 5). The rearrangement of arene oxides t o phenols occurs nonenzymatically and is termed the "NIH shift", since i t was discovered by workers at the National Institutes of Health (7),who worked out the mechanism. in a series of elegant deuterium labelling experiments. The NIH shift involves ionization of the epoxide, followed by a hydride shift and tautomerization of the intermediate dienone to the more stable phenol feq 6). Benzene is particularly toxic to hone marrow tissue. Toluene, a compound that has replaced henzene in many applications, is much less toxic. This difference may he due to differences in metaholism of the two. Whereas benzene is oxidized to the very electrophilic arene oxide, the major pathway in the metaholism of toluene is side-chain oxidation to give henzyl alcohol initially (a),and its subsequent oxidation to benzoic acid by other enzymes. In addition to oxidation a t carbon atoms, cytochrome P450 also catalyzes oxidations at heteroatoms. Tertiary amines are oxidized to N oxides, as shown for trimethylamine (eq 7). Secondary and some primary amines are oxi-
Figure 1. Oxidations.
Figure 2. RedUCliOnS.
dized to hydroxylamines (eq 8). Sulfides can undergo oxidation to yield sulfoxides, as is illustrated for the sedative1 tranquilizer chlorpromazine (eq 9). The second type of phase I reaction is reduction. Reduction is a much less common metabolic pathway than oxidation, hut it is important in the conversion of aromtic nitro and azo compounds to amines. Two examples of enzymatic reduction processes are shown in Figure 2. Reduction of nitro groups is effected by a number of different nitro reductases. In the reduction of nitro groups to amino groups (eq 10) nitroso compounds and hydroxylamines are intermediates. The best known example of azo reduction is the metabolism of Prontosil which yields (via a hydrazo intermediate) sulfanilamide (eq l l ) , the first of the sulfonamide antibiotics. The third major type of phase I reaction is enzymatic hydrolysis. Hydrolysis reactions of esters and amides are catalyzed by esterases and amidases that are found in the blood, liver, and many other tissues. Generally one finds that the rates of hvdrolvsis are what is to he exoected on the basis of ordinary sieric and electronic effects dn the reactivity of the carbonvl arouo. Thus, esters are usunllv hvdrolvzed faster than amidis. A-well-known example of ester hydrolysis is the metabolic conversion of acetylsalicylic acid (aspirin) to salicylic acid (Fig. 3, eq 12) in the liver. Steric effects can have a great effect on the hydrolysis rates of esters and amides. One pathway for deactivation of lidocaine, a local anesthetic, is by amide hydrolysis (eq 13) to give 2,5-dimethylaniline and N,N-diethylglycine. However, the unmethylated analogue of lidocaine hydrolyzes about 100 times faster than lidocaine itself, too fast for it to be a clinically useful anesthetic. Epoxides are hydrolyzed to 1,2-diols by the enzvme enoxide hvdrase. An alternate oathwav for the metaboiism i f henzene is the rytochrome'~-450iatnlgzed oxidation to henlene oxidp, followed by epoxidr hydrnse mediated
Figure 3. Hydrolyser.
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May 1967
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gAd +
PAPS
OH
0,
miOH
(16)
osoj
Figure 4. Conjugations Figure 5. Metabolism of benrapyrene.
conversion t o cyclohexa-1,3-diene-5,6-diol, which then dehydrates to form phenol (eq 14). The terms phase I1 reaction, conjugation, and synthesis are used more or less interchangeably to describe reactions in which a polar moiety, often derived from a carbohydrate or protein, is added to a xenobiotic or its phase I metabolite. Chemically these reactions are properly classified as functional group transfers and usually involve an activated intermediate that serves to facilitate the reaction. Conjugation reactions occur primarily with hydroxyl, amino, carboxyl, and sulfhydryl groups, which, if not already existing in the xenobiotic, were introduced or unmasked in phase I reactions. Only a few of the more common conjugation pathways are discussed here. Glucuronide formation is the most common conjugation pathway in mammals. Alcohols, carboxylic acids, and, less frequently, amines and thiols form glucuronides by reaction with uridine diphosphate glucuronic acid (UDPGA), which, in turn, is synthesized from glucose-1-phosphate. The reaction between UDPGA and the substrate xenobiotic is catalyzed by glucuronyl transferase, a microsomal enzyme present in the liver. An example of glucuronide formation is seen in the reaction of nhenol to form nhenvlelucuronide . (Fig. 4, eq 15). Sulfate conjugation is less common than glucuronide formation because the total sulfate pool is limited and can be readily exhausted by sulfoconjugation of endogenous molecules. The formation of sulfate conjugates occurs mainly with alcohols, phenols, and aromatic amines and involves three steps: activation of sulfate with adenosine triphosphate (ATP) to form adenosine-5'-phosphosulfate (APS), a second activation to form 3'-phosphoadenosine-5'-phosphosulfate (PAPS), and finally sulfate transfer to the substrate catalyzed by a sulfotransferase. Sulfate conjugation is illustrated by the reaction of p-hydroxyacetanilide (eq 16). Acylation is a common phase I1 reaction in which carboxylic acids are conjugated with endogenous polar amino acids such as elvcine or elutamine. or less commonlv. acid. .. . .. asoartic . serine, ornithine or taurine. In contrast to glucuronidation and sulf~,conjugation,here it is the xenobiotic substrate that isactiva~ed.The carhoxylic acid reacts with coenzyme A and ATP LO form an acvl coenwme A intermediate. The subsequent conjugation-with t& amino acid is catalyzed by a specific N-acyltransferase. The conversion of benzoic acid to its glycine conjugate hippuric acid (eq 17) has been known for many years. Some electrophilic substrates such as alkyl halides and epoxides form mercapturic acid conjugates by reaction with the tripeptide glutathione (GSH), which contains a nucleo-
--
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Journal of Chemical Education
philic sulfhydryl group and is found in concentrations up to 10 mM in the cytoplasm of liver and kidney cells. The glutathione conjugate then undergoes hydrolysis of the peptide bonds to remove two of the amino acid residues, glutamic acid and glycine. The 8-substituted cysteine residue is then acetylated on the nitrogen to give a mercapturic acid. Formation of a mercapturic acid is seen in the metabolism of naphthalene oxide (eq 18). The preceding discussion of the metabolic pathways of xenobiotic chemicals describes only a small fraction of the known reactions. The interested reader is referred elsewhere for a more complete discussion of such biotransformations (9-11). The examples discussed here are meant to illustrate the general strategy of the detoxication system-to introduce polar functionality into the xenohiotic molecule so that it might be more easily excreted. The system is very efficient. However, a small percentage of the time a "mistake" is made and the detoxication system inadvertently becomes a toxication system when reactive intermediates (usually electrophiles or free radicals) are created in phase I reactions, and rather than undergoing conjugation reactions, they escape the detoxication system to react with essential cellular molecules. Two examples will serve t o illustrate. Benzo(a)pyrene (BP) is a polycyclic aromatic hydrocarbon. BP is a potent carcinogen and is present in tobacco smoke. There are many metabolites of BP which have been isolated and identified, a few of which are shown in Figure 5. One metabolic pathway leads to the formation of the 7,sepoxide in a cytochrome P-450 catalyzed oxidation. This epoxide is hydrolyzed by epoxide hydrase to give the 7,8dihydrodiol. The 9,10 double bond then undergoes epoxidation to give a diolepoxide. I t is this "hay region" (12)epoxide that is helieved to be the ultimate carcinogen. This diol epoxide is apoor substrate for epoxide hydrase and glutathione-S-transferase, and therefore undergoes phase I1 conjugation very slowly. The reactive diol epoxide can then react w ~ t hendogenous cellular nucleophiles including DNA. The major covalent DNA adduct (-80%) occurs from the attack of the amino group of a guanine a t the benzylic carbon of the diol epoxide. Acetaminophen (p-hydroxyacetanilide) is a widely used mild analgesic and is readily available over the counter. Yet, a t high doses acetamino~hencauses liver and kidnev damage,which has resulted i i death in several cases. One &oposa1 (13) for the heoatotoxic oronerties of acetamino~henis shown in Figure 6:~here areseveral junctures in its metabolism where acetaminophen can be safely detoxified. Acet-
I
UDPCA
Figure 6. Metabolism of acetaminophen.
aminophen itself can undergo conjugation to form an Oglucuronide by reaction with UDPGA or a sulfate by reaction with PAPS (eq 16). Acetaminophen can also undergo cytochrome P-450 catalyzed N hydroxylation to give a hydroxamic acid which subseauentlv dehvdrates to eive a quinoneimine. This quinone
