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Chapter 33
Biotransformations Leading to Toxic
Metabolites: Chemical Aspect
Anne-Christine Macherey and Patrick M. Dansette
I. HISTORICAL BACKGROUND
II. INTRODUCTION
III. REACTIONS INVOLVED
IN THE BIOACTIVATION
PROCESS
A. Oxidation
B. Oxidative stress
C. Reduction
D. Substitutions: hydrolysis and
conjugation
E. Eliminations
F. Further biotransformations
leading to the ultimate
toxicant
IV. EXAMPLES OF
METABOLIC CONVERSIONS
LEADING TO TOXIC
METABOLITES
A. Acetaminophen
B. Tienilic acid
C. Halothane
D. Valproic acid
E. Troglitazone
V. CONCLUSION
REFERENCES
“La matière demeure et la forme se perd.”
“The matter remains and the form is lost.”
Pierre de Ronsard
I. HISTORICAL BACKGROUND
As drugs are usually foreign chemicals, history of
concern for the biotransformations of drugs leading to toxic
metabolites formation is intrinsically linked to the history
of xenobiotic metabolism studies. The International Society
for the Study of Xenobiotics (ISSX) website (http://www.
issx.org) presents an overview of the field history where
some key figures may be pointed out.
One is probably Richard Tecwyn Williams who introduced the Phase I and II classification of xenobiotics metabolism reactions. Although his emblematic book1 was called
“Detoxication mechanisms,” he estimated that, in some
cases, metabolism may increase toxicity. He also considered
that this “bioactivation” may occur during the Phase II reactions (usually considered as detoxication reactions), and not
only that of Phase I (functionalization reactions).
Quite at the same time, Bernard Brodie studied the
antimalarial atabrine (quinacrine) metabolism in order to
Wermuth’s The Practice of Medicinal Chemistry
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avoid the toxic side effects of the drug. He also developed
some new analytical methodologies, necessary for metabolic studies. Then he put together a group of researchers
(including Julius Axelrod, James Gillette and many others)
in this field, and they published many studies of great importance related to drug metabolism, most famous probably
concerning acetaminophen. Among these works, these
scientists developed the covalent binding theory concept,
which provides an explanation for the toxic side effects of
drugs. Following the work of James and Elizabeth Miller
on covalent binding of polycyclic aromatic hydrocarbon
electrophilic metabolites on DNA in the 1940s, Brodie
et al. suggested that in vivo bioactivation may lead to the
formation of electrophilic entities, which are capable of
linking with biological macromolecules, thus inducing disturbances in cellular functions.
The discovery of mixed function oxidases during
the 1950s and the characterization of cytochrome P450
by Omura and Sato2 were a “revolution” in the field of
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All rights reserved.
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II. Introduction
xenobiotic metabolism. Remmer discovered that cytochrome P450 may be induced by phenobarbital, and Conney
characterized the induction with 3-methyl cholanthrene.
These works3 were of great importance for mechanistic
studies of drug metabolism. A new step was done in 1999
with the first crystallization of a mammalian cytochrome
P450 by Johnson, which provided new perspectives in safer
drug design.
Induction of cytochrome P450 synthesis suggests that
xenobiotics may exert an effect on the genome: the use of
genomics and proteomics represents a new challenge for
predictive toxicology in drug design.
FIGURE 33.1
Indirect toxicity.
II. INTRODUCTION
Toxicity is the result of the more or less harmful action of
chemicals on a living organism. Toxicology, the study of
toxicity, is situated at the border of chemistry, biology, and
in some cases, physics. Molecular toxicology tries to elucidate the mechanisms by which chemicals exert their toxic
effects. Because many foreign chemicals enter the body
in inert but unexcretable forms, biotransformations are an
important aspect of the fate of xenobiotics.4,5 In the case
of drugs, metabolic conversions may be required for therapeutic effect (“prodrugs”; see Chapter 36 for a detailed discussion of prodrugs). In other cases, metabolism results in
a loss of the biological activity. Sometimes, biotransformations produce toxic metabolites. The last process is called
toxification or bioactivation. It should be emphasized that
the general principles of pharmacology embrace the occurrence of toxic events: although biotransformation processes
are often referred to as detoxification, the metabolic products
are, in a number of cases, more toxic than the parent compounds. For drugs, whether biotransformations lead to the
formation of toxic metabolites or to variations in therapeutic effects depends on intrinsic (such as the genetic polymorphism of some metabolism pathways) and extrinsic
(such as the dose, the route or the duration) factors. The
biochemical conversions are usually of an enzymatic nature
and yield reactive intermediates, which may be implicated
in the toxicity as far as the final metabolites. The primary
events, which constitute the beginning of the toxic effect
may result, after metabolism, from an inhibition of a specific (and in most cases enzymatic) cellular function, an
alkylating attack or an oxidative stress.
With regard to the toxicity arising from metabolites
(“indirect toxicity”), three cases may be distinguished
(Figure 33.1):
A. Biotransformation begins with the transient formation of
a reactive intermediate, whose lifetime is long enough to
allow an attack on cellular components. This occurs when
a reactive intermediate (generally radicals or electrophiles
such as a carbonium ion) is formed and reacts rapidly
with cellular macromolecules (such as unsaturated lipids,
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proteins, nucleic acids…), thus leading to their degradation and finally to cellular necrosis.
B. The first step of the metabolic process yields a primary
metabolite, which can, in some cases, accumulate in the
cell and react with cellular components before being
transformed.
C. The final metabolites, when in excess, may accumulate
and react with cellular macromolecules.
Usually, metabolic conversions are divided into two
major types of reactions (see chapter 32 for a detailed discussion of metabolic biotransformations). Phase I reactions,
or functionalization reactions, involve the introduction of a
polar functionality such as a hydroxyl group into the xenobiotic structure. During Phase II reactions, this group is
subsequently coupled (or conjugated) with an endogenous
cofactor, which contains a functional group that is usually
ionized at physiological pH. This ionic functional group
facilitates active excretion into the urinary and/or hepatobiliary system. The elimination by transport mechanism is
sometimes also called “Phase III.”
Because bioactivation is mainly an activation of xenobiotics to electrophilic forms, which are entities capable
of reacting irreversibly with tissue nucleophiles, biotransformations leading to toxic metabolites are in most cases
Phase I reactions. But Phase II reactions may also give rise
to toxic phenomena, for example, when conjugation produces a toxic metabolite, or when it is responsible for a
specific target organ toxicity by acting as a delivery form
to particular sites in the body where it is hydrolyzed and
exerts a localized effect. Also, the final toxic metabolite
may be formed by combinations of several Phase I and
Phase II reactions. Because of the increasing understanding
of drug metabolizing enzymes, some authors6 claim that
Williams “Phases I and II” classification is now inaccurate
and even misleading. Pointing out the fact that Williams
only introduced the classification at the end of his book and
did not use it in his monograph, they consider it would be
now wiser to avoid using any special category.
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CHAPTER 33 Biotransformations Leading to Toxic Metabolites: Chemical Aspect
III. REACTIONS INVOLVED IN THE
BIOACTIVATION PROCESSES
During the biotransformations affecting xenobiotics, five
major kinds of chemical reactions may occur: oxidations
(by far the most important), reductions, hydrolysis, substitutions, and eliminations. As Phase I and II reactions are
part of this classification, each class of reactions can give
rise to toxic metabolites.
A. Oxidation
Several enzymatic systems are involved during the oxidative transformations of xenobiotics. Whether substances
act upon one enzyme rather than another depends not only
on its specific function, but also on the electromolecular
environment. The most important is the microsomal drug
metabolizing system known as cytochrome P450 (CYP)
monooxygenase, which is localized mainly in the liver
and is involved in most biological oxidations of xenobiotics.7–9 Those include C-, N- and S-oxidations, N-, O- and
S-dealkylation, deaminations, and certain dehalogenations.
Under anaerobic conditions, it can also catalyze reductive
reactions. The CYP monooxygenase system is a multienzymatic complex constituted by the CYP hemoprotein,
the flavoprotein enzyme NADPH CYP reductase, and the
unsaturated phospholipid phosphatidylcholine. The isoforms involved in xenobiotic metabolism are membrane
bound enzymes situated in the endoplasmic reticulum. After
FIGURE 33.2 Catalytic cycle of cytochrome P450 (CYP)
monooxygenase.
cell lysis for in vitro studies, they are found in the microsomal fraction. There are numerous isoforms (more than
6,000 known in all species). Thus a nomenclature based on
their sequence similarity has been designed, and they are
classified in families and subfamilies: for instance CYP3A4
is the major human CYP, CYP is for cytochrome P450,
3 for the number of the family (more than 40% sequence
identity), A for the letter of the subfamily (more than 55%
sequence identity) and 4 the number in the subfamily. The
human genome shows 57 complete CYP sequences plus a
number of pseudo-genes. The CYPs involved in xenobiotic
metabolism9 (about 15) belong to families 1 to 4. The catalytic mechanism of CYP involves a formal (FeO)3⫹ complex formed by the elimination of H2O from the iron site
after two electrons have been added (Figure 33.2).
Another oxidative enzyme is the FAD-containing
monooxygenase, which is capable of oxidizing nucleophilic
nitrogen, sulfur, and organophosphorus compounds. The
flavoprotein binds NADPH, oxygen and then the substrate.
The oxidized metabolite is released, followed by NADP.
Alcohol dehydrogenase and aldehyde dehydrogenase catalyze the oxidation of a variety of alcohols and aldehydes
into aldehydes and acids, respectively, in the liver. Xanthine
oxidase oxidizes several purine derivatives such as theophylline. Monoamine oxidase (MAO) and diamine oxidase
convert amines into alkyl or aryl aldehydes by oxidation of
the amine to an imine followed by subsequent hydrolysis.
Peroxidases are oxidative enzymes, which couple the reduction of hydrogen peroxide and lipid hydroperoxides to the
oxidation of other substrates. This co-oxidation is responsible for the production of reactive electrophiles from aromatic
amines (e.g. the highly carcinogenic benzidine), phenols,
hydroquinones, polycyclic aromatic hydrocarbons, etc.
The oxidation reactions can be described in terms of a
rather common chemistry that involves the abstraction of
either a hydrogen atom or a nonbonded (or π) electron by
the iron-oxo porphyrin complex (Figure 33.3). The highvalent complex electronic configuration is unknown, but is
usually written as FeV—
—O.
The one-electron oxidation yields transient radicals
(Figure 33.4), which are transformed into more stable forms.
These radicals can incorporate an oxygen atom by
abstraction of a hydroxyl group from the CYP iron-oxo species. This yields an oxidized derivative that may be sometimes more toxic than the parent compound or susceptible
to further metabolic conversions. Free radicals may also
FIGURE 33.3 CYP oxidation process.
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III. Reactions Involved in the Bioactivation Process
bind to the site of their formation, thus leading to inhibition
or inactivation of the enzyme. When the radical is not efficiently controlled by the iron, it may leave the active site.
The subsequent released radical is able to produce damage
to unsaturated fatty acids, thus leading to lipid peroxidation
and destruction of the cellular structure. Another mode of
the radical stabilization is a second one-electron oxidation,
which consists of the loss of another electron. The fate of
free radicals is now extensively studied because of their
great capacities for forming covalent bonds with cellular
macromolecules.10–12
Tertiary amines containing at least one hydrogen on the
α carbon may either be N-oxidized (leading to an N-oxide in
the case of tertiary amines), or C-oxidized, thus leading to a
carbinolamine. The latter, usually being unstable, splits into
a secondary amine and an aldehyde moiety (Figure 33.8).
Several electron transfer mechanisms have been proposed.7–9
During the oxidation of nitrosamines, the hydroxylated
derivative formed cleaves spontaneously into highly reactive
metabolites capable of alkylating nucleophilic sites in the
cellular components.
1. C-H bond oxidations
These oxidations, which are usually catalyzed by CYP
monooxygenases, produce hydroxylated derivatives.13 When
the C-H bond is located in the α position to a heteroatom
(such as O, S, N, halogen), the α hydroxylated derivative
obtained is usually unstable and may be further oxidized or
cleaved (Figure 33.5).
The antibiotic chloramphenicol is oxidized by CYP
monooxygenase to chloramphenicol oxamyl chloride
formed by the oxidation of the dichloromethyl moiety of
chloramphenicol followed by elimination of hydrochloric
acid14 (Figure 33.6). The reactive metabolite reacts with the
␧-amino group of a lysine residue in CYP15 and inhibits the
enzymatic reaction progressively with time. This type of
inhibition is a time-dependent inhibition or a mechanismbased inhibition or inactivation, and the substrate involved
historically has been called a suicide substrate because
the enzymatic reaction yields a reactive metabolite, which
destroys the enzyme.16
In the case of chloroform, the unstable trichloromethanol loses hydrochloric acid and forms phosgene, which is
very reactive (Figure 33.7).17
FIGURE 33.6 Metabolic activation of chloramphenicol.
FIGURE 33.4
One-electron oxidation.
FIGURE 33.5
C-H bond oxidation in the α-position to a heteroatom.
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FIGURE 33.7
Oxidation of chloroform.
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CHAPTER 33 Biotransformations Leading to Toxic Metabolites: Chemical Aspect
does not exhibit mutagenic or carcinogenic activity, but
reacts nonenzymatically with liver proteins and produces
hepatic necrosis.20 The isomeric 2,3-epoxide rearranges
very quickly to 2-bromophenol and is less toxic. A secondary CYP-catalyzed oxidation to hydroquinone and
benzoquinone also can occur. In this alternative pathway,
conjugation with glutathione can lead to the formation of
products, which may elicit their toxicity elsewhere than the
liver and especially in the kidney.20
3. N-oxidations
FIGURE 33.8 Oxidation of a tertiary amine.
Tertiary amines are transformed into N-oxides (generally
less toxic), but primary and secondary amines are oxidized
into hydroxylated derivatives (hydroxylamines). This oxidation is responsible for the hepatotoxicity and mutagenicity of acetamino-2-fluorene (Figure 33.11).21
Nitrenium ions may occur during bioactivation of aromatic amines and amides, which are usually N-oxidized
into N-hydroxylated derivatives. By sulfation or esterification followed by elimination of the newly formed leaving
group, the latter may be transformed into highly reactive
nitrenium ions. In the case of aromatic nitrenium ions they
are in equilibrium with their tautomeric aromatic carbocations, which react with cellular nucleophilic macromolecules
(nucleic acids, etc.).
4. Heteroatom oxidations
FIGURE 33.9
Oxidation of aflatoxin B1.
2. Unsaturated bond oxidations
Double bonds are oxidized by CYP monooxygenases into
epoxides, which are generally very reactive. Epoxides are
considered responsible for the toxicity of the unsaturated
compounds.
The hepatocarcinogenicity of aflatoxin B1 (AFB1) is
known to be due to the epoxide (AFB1-oxides) formed,
which binds directly with the N-7 atom of a guanine
molecule in DNA (Figure 33.9).18
Aromatic chemicals are metabolized into unstable areneoxides, which, as epoxides, are comparable to potentially
equivalent electrophilic carbocations. These metabolites
react easily with thiol groups derived from proteins, leading,
for example, to hepatotoxicity. Bromobenzene seems to target a large group of functionally diverse hepatic proteins, as
demonstrated recently in a proteomic analysis.19 The chemical is oxidized (Figure 33.10) into a 3,4-epoxide, which
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Heteroatoms such as nitrogen or sulfur are oxidized at their
nonbonded peripheric electrons as described for thiophene
(Figure 33.12a).22 Thiophene is oxidized to thiophene
sulfoxide, which is unstable and dimerizes spontaneously to
thiophene S-oxide dimers through a Diels–Alder reaction.23,24 They also react with nucleophiles like the thiol
group of glutathione or proteins, giving glutathione or protein adducts. In addition, thiophenes are oxidized to unstable thiophene epoxides, which rearrange spontaneously to
thiolenones as found recently for 2- and 3-phenylthiophenes
(Figure 33.12b). In fact, there is a competition between
S-oxidation (sulfoxide pathway) and double bond oxidation
(epoxide pathway). In the presence of glutathione, adducts
formed from both reactive intermediates have been found,
in addition to thiophene S-oxide dimers and the thiolenones
tautomers of hydroxythiophenes.25,26
Halogenated aromatic compounds may also be oxidized
by CYP monooxygenases, yielding hypervalent halogenated compounds.
B. Oxidative stress
Oxidative stress has been defined as a disturbance in the
pro-oxidant–antioxidant balance in favor of the pro-oxidant
state resulting from alterations in the redox state of the cell.
The stepwise reduction of oxygen into superoxide anion,
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III. Reactions Involved in the Bioactivation Process
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FIGURE 33.10 Metabolism of bromobenzene.
FIGURE 33.11 N-oxidation of acetamino2-fluorene.
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CHAPTER 33 Biotransformations Leading to Toxic Metabolites: Chemical Aspect
FIGURE 33.12 (a) oxidation of thiophene,
(b) oxidation of 2-phenylthiophene.
hydrogen peroxide, hydroxyl radical, and finally water,
which accounts for about 5% of the normal oxygen reduction (versus 95% by means of the mitochondrial electron
transport chain), may be increased by the redox cycling
of some xenobiotics such as quinones or nitro-aromatic
derivatives. These compounds are susceptible to one-electron
reduction, which yields radical structures that may be backoxidized to the parent compound. During this reoxidation,
oxygen is reduced into superoxide anion. The oxygen
reduction products are highly reactive entities that attack
all the cellular components, especially when their normal
degradation systems (superoxide dismutase, glutathione
peroxidase, catalase) are overburdened. The polyunsaturated
lipids are especially sensitive to these attacks because they
are susceptible to a membrane-degrading peroxidation.
C. Reduction
Reductive biotransformations of several compounds such
as polyhalogenated, keto, nitro and azo derivatives, are
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catalyzed by a variety of enzymes that differ according to the
substrates and the species. The liver CYP-dependent drug
metabolizing system is capable of reducing N-oxide, nitro
and azo bonds, whereas the cytosolic nitrobenzene reductase
activity is mainly due to CYP reductase, which transforms
nitrobenzene into its hydroxylamino derivative. NADPH
cytochrome c reductase is also able to catalyze the reduction
of nitro compounds. These metabolic conversions may also
be brought about by gastrointestinal anaerobic bacteria.
Reductive processes that occur during the metabolism
of xenobiotics involve either one-electron reduction or a
two-electron transfer.
Ionic reduction using a hydride occurs in vivo during the
reduction catalyzed by NADH or NADPH enzymes, whereas
one-electron reduction releases a radical structure, which
may contribute to the toxic effect. Figure 33.13 illustrates
the biotransformations affecting the anthracycline antitumor
drug daunomycin.27 Recent studies suggest that nitric oxide
synthases may contribute to the cardiotoxicity, probably
because of their structural similarities with CYP reductase.28
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III. Reactions Involved in the Bioactivation Process
FIGURE 33.13 Biotransformations
of daunorubicin.
1. Reduction of polyhalogenated compounds
2. Reduction of nitro compounds
Some polyhalogenated compounds, such as CCl4, BrCCl3
and halothane (CF3-CHBrCl), when in the presence of the
reduced form of CYP, may undergo one-electron reduction29,30 (Figure 33.14), which leads to a radical that may
be transformed by different pathways.
The radical formed may add directly on the unsaturated
lipid bonds or initiate an unsaturated lipid peroxidation or
undergo another one-electron reduction. The last reaction
yields a carbene that can form a complex with the iron of
the reduced form of CYP. Reduction of polyhalogenated
compounds gives rise to several reactive intermediates,
such as radicals, carbenes and peroxides, whose participation in the toxic effect varies greatly.13
The different steps of the biotransformations that produce
a primary amine from an aromatic nitro compound involve a
nitro radical-anion, a nitroso derivative, a nitroxyl radical,
a hydroxylamine and then the primary amine (Figure 33.15).
Each of these different intermediates may contribute
to the toxicity. Hydroxylamines are often responsible for
methemoglobinemia,31 whereas mutagenic and carcinogenic
activity may be due to the combination of nitro radical-anion,
nitroso derivatives or esterified hydroxylamine (such as
sulfate derivatives) with cellular macromolecules.
Carcinogenicity may also be the result of the oxidative
stress subsequent to the formation of oxygen–reduction products (superoxide anion, hydrogen peroxide, hydroxyl radical)
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CHAPTER 33 Biotransformations Leading to Toxic Metabolites: Chemical Aspect
FIGURE 33.14 Reduction of polyhalogenated
compounds.
FIGURE 33.15 Reductive biotransformation
of nitro arene compounds.
during redox cycling of the nitro radical-anion, which restores
the parent nitro compound.
D. Substitutions: hydrolysis and
conjugation
3. Reduction of azo compounds
Among substitution reactions, ester and amide hydrolysis are common, and often operate during detoxification
processes. Both specific enzymatic and chemical hydrolysis may occur. Acid-catalyzed reactions may occur in the
stomach and the kidney, whereas base-catalyzed reactions
may be assisted by the alkaline pH of the intestine.
Azo compounds are susceptible to reduction, first to hydrazo
intermediates, which are reductively cleaved into the appropriate amines. It has been proposed32 that the first step, as with
nitro compounds, is the formation of an azo-anion radical.
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III. Reactions Involved in the Bioactivation Process
Phase II, or conjugation reactions, are also substitution
reactions, which proceed by means of an endogenous and
generally activated electrophile. In mammals, five major
conjugation reactions of xenobiotics exist and are mediated
by transferase enzymes. Acid compounds, through their
acyl-CoA ester, may also be conjugated with amino acids
such as glycine, glutamine, and taurine. The specificity for
the endogenous agent is high, but the specificity for the
xenobiotic is broader.
To a great extent, conjugation produces excretable and
nontoxic metabolites and thus is referred to as detoxification,
but exceptions exist in each class of conjugation reaction.
A more-in-depth discussion of Phase II metabolism can be
found in Chapter 32.
1. Glucuronic acid conjugation
This substitution involves the transfer of a glucuronic acid
from uridine diphosphate glucuronic acid (UDPGA) to
a functional group in the xenobiotic substrate. The group
may be a hydroxyl, carboxylic acid, amino or sulfur
functional group. Glucuronides are never directly implicated in toxicity but are sometimes responsible for targetorgan toxicity. Aromatic amines may be converted in
the liver into hydroxylamine O-glucuronides, which are
excreted in the urine and broken down in the bladder (if
its pH is acidic) to liberate the proximate hydroxylamine
carcinogen.
2. Sulfation
Sulfate conjugation gives a polar and ionized conjugate by
means of the esterification of a hydroxyl group with sulfate
ion (transferred from 3⬘-phosphoadenosine-5⬘-phosphosulfate or PAPS). The reaction is catalyzed by a hydrosoluble
sulfotransferase. Sulfation sometimes gives rise to reactive
intermediates that may undergo further reactions to yield
electrophilic metabolites. In the case of 2-acetaminofluorene, the O-sulfate moiety is a facile leaving group, and
this cleavage produces nitrenium ions, which act as alkylating
agents for DNA (Figure 33.11).
3. Acetylation
Acetylation is a very common metabolic reaction, which occurs
with amino, hydroxyl or sulfhydryl groups. The acetyl group
is transferred from acetyl-Coenzyme A, and the reaction is catalyzed by acetyltransferases. An important aspect of this kind
of substitution is the genetic polymorphism of one acetyltransferase in humans, who are divided into fast and slow acetylators. In a few cases, the conjugates are further metabolized
to toxic compounds, as is seen with isoniazid. Some evidence
exists that acetylation of the antitubercular isoniazid leads to
enhanced hepatotoxicity of the drug.33,34 Acetylation followed
by hydrolysis and CYP-dependent oxidation yields free acetyl
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radicals35 or acylium cations, which may acetylate the nucleophilic macromolecule functions (Figure 33.16).
4. Glutathione conjugation
Substitution reactions of xenobiotics with glutathione are the
most important and contribute efficiently to detoxification.
Nevertheless, in some cases, such as vicinal dihalogenated
compounds, glutathione conjugation produces monosubstituted derivatives, which may cyclize into a highly electrophilic episulfonium ion (Figure 33.17).36
5. Methylation
Methylation is rarely of quantitative importance in the
metabolism of xenobiotics. The methyl group is transferred
from the nucleotide S-adenosyl-l-methionine (SAM) by
means of a methyltransferase. The functional groups that
undergo methylation include primary, secondary and tertiary
amines, pyridines, phenols, catechols, thiophenols. The azaheterocycle pyridine is metabolized to the N-methylpyridinium ion, which is more toxic than pyridine itself37 (Figure
33.18). The binding properties of the ionized metabolite are
disturbed by the loss of its hydrophobic feature, resulting
from the polarity inversion.
E. Eliminations
Eliminations of hydrogen and a halogen occur sometimes
during the metabolism of halogenated xenobiotics and
lead to an alkene. The double bond may be oxidized into
an epoxide by means of oxidative enzyme systems as discussed above. Dehydrogenation, dehydrochlorination and
dechlorination are (with oxidation) the different metabolic
pathways of the γ-isomer of the insecticide hexachlorocyclohexane (lindane).38
F. Further biotransformations leading to
the ultimate toxicant
Other reactions must be mentioned beside the major reactions described above. These reactions may be responsible
for the transformation of a toxic metabolite into the ultimate toxicant.39 Rearrangements and cyclizations are examples of reactions involved in these processes. In the case of
the solvent hexane (Figure 33.19), the toxic metabolite,
2, 5-hexanedione, is formed by four successive oxidations
of the molecule. The condensation of the γ-dicetone with
the lysyl amino group of a neurofilament protein is followed
by a Paal–Knorr cyclization reaction. This is the initial process that explains the hexane-induced neurotoxicity.40 A further auto-oxidation of the N-pyrrolyl derivatives leads to the
cross-linking of the axonal intermediate filament proteins
and the subsequent occurrence of peripheral neurotoxicity.41
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CHAPTER 33 Biotransformations Leading to Toxic Metabolites: Chemical Aspect
FIGURE 33.16 Bioactivation of isoniazid.
FIGURE 33.18
FIGURE 33.17
Bioactivation to episulfonium ion.
Analogous pyrrolyl derivatives are also found as
furan metabolites. Furans are oxidized by CYP to reactive furan-epoxides, which rearrange to ene-dial or eneketo-aldehyde metabolites (Figure 33.20).26,42,43 After
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Bioactivation of pyridine.
reaction with thiols and amines like lysine, they form
stable pyrrolic derivatives. This first depletes the cell
of glutathione then creates cross-links in proteins and
toxicity.
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IV. Examples of Metabolic Conversions Leading to Toxic Metabolites
FIGURE 33.19
Bioactivation of hexane.
IV. EXAMPLES OF METABOLIC
CONVERSIONS LEADING TO
TOXIC METABOLITES
The formation of toxic metabolites and/or intermediates during
the metabolism of drugs may occur by a considerable variety
of pathways that are mediated by several enzyme systems.
The following five examples do not represent an exhaustive
list of the bioactivation processes, but are samples of original,
significant and/or well-known drugs whose biotransformations lead to toxic compounds by the main types of reactions
discussed above. Two of them (acetaminophen and tienilic
acid) are CYP-mediated oxidations. Halothane acts through
both oxidative and reductive biotransformations. Valproic
acid is toxic through its elimination product. The toxicity of
troglitazone seems to involve two distinct metabolic pathways, leading to both alkylating and oxidative stresses.
FIGURE 33.20
Bioactivation of furans.
A. Acetaminophen
The analgesic acetaminophen (4-hydroxyacetanilide, paracetamol) exhibits hepatotoxicity when administered in
very high doses (approximately 250 mg/kg in rat and about
13 g for a 75 kg human ).44 The metabolite responsible is
known to be the N-acetyl-p-benzoquinone imine (NAPQI)
(Figure 33.21).45
The formation of NAPQI may proceed via CYP2E1,46 but
also via peroxidases such as prostaglandin hydroperoxidase.
The most commonly described mechanism proposes that
metabolic activation occurs through N-oxidation of acetaminophen to N-hydroxyacetaminophen followed by dehydration to NAPQI (Figure 33.22).47
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However, it seems that N-hydroxyacetaminophen is not
a major intermediate in the oxidation of acetaminophen.
The formation of NAPQI probably proceeds by two
successive one-electron oxidations48 (Figure 33.23).
During the first step, a one-electron oxidation yields a
phenoxy radical (Ar-O•).49 The presence of the radical was
supported by fast flow ESR spectroscopy in the presence of
horseradish peroxidase. In the second one-electron oxidation,
the phenoxy radical is oxidized to NAPQI. As described in
Figure 33.21, the highly electrophilic NAPQI may easily
react with glutathione or protein thiol groups according to
a Michael-type addition. The attack of liver protein thiol
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CHAPTER 33 Biotransformations Leading to Toxic Metabolites: Chemical Aspect
FIGURE 33.21 Biotransformation pathway of
acetaminophen.
FIGURE 33.22 Oxidation of acetaminophen according to
the “N-hydroxyacetaminophen pathway.”
groups and the subsequent adduct formation is frequently
mentioned in the mechanism of acetaminophen hepatotoxicity.
In mice, a number of proteins were identified such as glyceraldehyde-3-phosphate dehydrogenase,50 calreticulin and
the thiol: protein disulfide reductases Q1 and Q551 and this
number is increasing with the advances of proteomics.52
Another hypothesis for the mechanism of toxicity is
supported by the oxidative potency of NAPQI, but still
suffers from lack of evidence.53 NAPQI is a good oxidant
Ch33-P374194.indd 686
for thiols of cellular components and pyridine nucleotides.
Moreover, it may undergo a redox cycling with formation
of superoxide anion by means of an oxygen one-electron
reduction (Figure 33.24).
The stepwise reduction of oxygen produces hydrogen
peroxide, and finally, a hydroxyl radical, which is a strong
oxidant implicated in cellular oxidative stress. This oxidative
stress causes glutathione depletion, a disruption of the cellular
calcium regulation and modifications of cellular proteins, thus
5/30/2008 6:35:33 PM
IV. Examples of Metabolic Conversions Leading to Toxic Metabolites
FIGURE 33.23
radical.
Oxidation of acetaminophen by means of the phenoxy
FIGURE 33.24
Redox cycling of N-acetyl-p-benzoquinone imine.
leading to cell death. Some biochemical parameters related
to necrotic and apoptotic processes are affected in acetaminophen-exposed PC12 cells transfected with CYP2E1.54,55
It therefore appears that both covalent (e.g. alkylation) and
noncovalent (e.g. oxidative stress) interactions play a major
role in the pathogenesis of acute lethal cell injury caused by
NAPQI.56 At present, it is not possible to identify which of
these two interactions is the critical event in initiating acetaminophen hepatotoxicity, even if some authors suggest that
the characteristic features of oxidative stress are more likely
the consequences of damage mediated by protein adduction.57
B. Tienilic acid
Tienilic acid is a uricosuric diuretic drug that may cause
immunoallergic hepatitis in 1 in 10,000 patients, a side
effect that resulted in its withdrawal from the market. The
Ch33-P374194.indd 687
687
immunoallergic hepatitis was associated with the appearance
of circulating antireticulum antibodies called anti-LKM2
antibodies, which are directed toward a liver endoplasmic
reticulum protein.58,59 From these observations, the mechanism of the immunotoxicity associated with the prolonged
use of tienilic acid was elucidated by the Mansuy group.60–62
Tienilic acid is oxidized in the liver by CYP monooxygenase to 5-hydroxytienilic acid, which is the major urinary
metabolite (about 50% in human). In humans, the bioactivation of tienilic acid depends on CYP2C9. This isoform
is one of the major forms of CYP in the human liver. This
oxidation occurs through an electrophilic intermediate
capable of alkylating very specifically the CYP61,63,64 leading
to its inactivation. This mechanism-based inactivation is
also observed with many xenobiotics such as alkenes with
terminal unsaturation, alkynes, strained cycloalkylamines,
4-alkyldihydropyridines, benzodioxoles, and some tertiary
amines.16,65 The irreversible binding of the compound with
CYP leads to an immune response and to generation of
antibodies against both the modified protein and its native
form. In fact, the autoantibodies anti-LKM2 present in
hepatitis patients recognize CYP2C9 both as native protein and as modified protein. In addition, patient sera contain antibodies to tienilic acid-modified proteins. It has also
been demonstrated in a rat model that tienilic acid modified
CYP2C9 is exported to the plasma membrane of hepatocytes66 and has been shown that tienilic acid treated rabbit
hepatocytes, when first incubated with anti-LKM2, were
lysed by human NK cells.67 Thus, it is hypothesized that
appearance of tienilic acid bound proteins on the hepatocyte surface triggers their cytolysis.
In the case of tienilic acid, the electrophilic reactive species is unknown. This is either a thiophene sulfoxide, as has
been demonstrated for its 3-isomer,68 or a thiophene epoxide
(Figure 33.25). In both cases the electrophilic character of
the intermediate is enhanced by the presence of an activating 2-keto group. In any event, this electrophilic species
reacts with the enzyme CYP2C9 where it is produced and
inactivates it efficiently (one inactivation event every 13
turnover).61 The covalent binding of tienilic acid to CYP2C9
has been directly observed by mass spectrometry.69 This
reaction occurs in all patients with active CYP2C9 using
this drug; however, very few produce anti-LKM2 and have
hepatitis, which suggests some specificity in their immune
response.
C. Halothane
Halothane is a widely used anesthetic drug that occasionally results in severe hepatitis. About 60–80% of the
dose is eliminated in unmetabolized form during the 24 h
following administration to patients. This compound is
metabolized in the presence of CYP monooxygenase
CYP2E1 according to the two main pathways13 depicted in
Figure 33.26.
5/30/2008 6:35:34 PM
688
CHAPTER 33 Biotransformations Leading to Toxic Metabolites: Chemical Aspect
FIGURE 33.25 Tienilic acid biotransformation to reactive intermediates
and stable metabolites.
The major biotransformation pathway involves an
oxidative step with introduction of an oxygen atom and
subsequent formation of halohydrin. The unstable halohydrin loses hydrobromic acid to yield trifluoroacetyl chloride,
which in turn is hydrolyzed to trifluoroacetic acid. This
final metabolite is found in the urine.70
In conditions of low levels of oxygen, a reductive pathway (10%) is enhanced and yields a free radical intermediate characterized as 1-chloro-2,2,2-trifluoroethyl radical.
Another one-electron reduction produces the 1-chloro-2,2,2trifluoroethyl carbanion, which may undergo two possible
kinds of eliminations.
One is the abstraction of a fluoride ion according to a E1cB
elimination, which yields 1-chloro-2,2-difluoroethylene. This
metabolite is eliminated by exhalation. Early studies suggested
that a second elimination process might be an α-elimination
of a chloride ion, which produces trifluoromethylcarbene,71
but this was later reconsidered.72 It was hypothesized that a
Ch33-P374194.indd 688
carbene complex with the FeII in the active site might lead to
inactivation of the CYP, but this inactivation is now thought
to be due to the formation of an iron-σ-alkyl complex derived
from the 1-chloro-2,2,2-trifluoroethyl radical.
The initially formed 1-chloro-2,2,2-trifluoroethyl radical
may also cause a radical attack of polyunsaturated lipids,
which produces 1-chloro-2,2,2-trifluoroethane. This mechanism is similar to the pathway described with the trichloromethyl radical formed during the one-electron reduction
of carbon tetrachloride (Figure 33.14). The trichloromethyl
radical may initiate a peroxidation of unsaturated lipids from
the membrane with subsequent liberation of chloroform.
Several studies have demonstrated that halothane hepatotoxicity is mainly due to an immune reaction toward modified
proteins of the liver. In fact, these proteins are trifluoroacetylated on their ␧-NH2-lysyl residue by the trifluoroacetyl
chloride formed during the oxidative metabolism of
halothane.73,74 The product of the reaction can act as a foreign
5/30/2008 6:35:35 PM
IV. Examples of Metabolic Conversions Leading to Toxic Metabolites
689
FIGURE 33.26 The major metabolic pathways of halothane.
epitope, and the drug–protein conjugate, called neoantigen,
elicits an immune response toward the liver75 (Figure 33.27).
A related fluorocarbon used in air conditioning systems,
HCFC 1,2,3, is metabolized to the same acyl halide and
was recently implicated in an epidemic of liver disease in
nine workers of a Belgian factory.76 All patients had serum
antibodies to trifluoroacetylated proteins.
D. Valproic acid
Valproic acid is an anticonvulsant agent used for the
therapy of epilepsy, which occasionally results in hepatotoxicity in young children. The toxicity is characterized by
Ch33-P374194.indd 689
mitochondrial damage, impairment of fatty acid β-oxidation and lipid accumulation.
It has been proposed that hepatotoxicity is a consequence
of the further biotransformation of the valproic acid metabolite 2-propyl-4-pentenoic acid (also called Δ4VPA).77
As depicted in Figure 33.28, Δ4VPA is not formed by
dehydration of 4- or 5-hydroxy valproic acids, which are,
with the glucuronide conjugate, the major metabolites of
valproic acid.78 The mechanism is proposed to involve
an initial hydrogen abstraction to generate a transient free
radical intermediate. It has been demonstrated that the carbon-centred radical was localized at the C4 position. The
radical undergoes both recombination (which yields 4hydroxy valproic acid) and elimination (which produces
5/30/2008 6:35:36 PM
690
CHAPTER 33 Biotransformations Leading to Toxic Metabolites: Chemical Aspect
FIGURE 33.27 Biotransformation of halothane to
trifluoroacetyl chloride and the subsequent binding to
protein.
FIGURE 33.28 Bioactivation of valproic acid to
Δ4VPA.
the unsaturated derivative Δ4VPA). The formation of
these metabolites is catalyzed in rat by CYP4B179 and in
human by CYP2C9.9 Δ4VPA is a hepatotoxic and strong
teratogenic compound in animal models. In addition to that
metabolic pathway, valproic acid undergoes biotransformation leading to (E)Δ2VPA, which is devoid of embryotoxic
effect in rodents.80
Further biotransformations of Δ4VPA involve both
the liver microsomal CYP enzymes and the fatty acid
β-oxidation pathway (Figure 33.29). The mixed-functionoxidase system metabolizes the unsaturated metabolite to a
γ-butyrolactone81 derivative through a chemically reactive
entity that is a mechanism-based inhibitor of CYP. The
alkylation of the prosthetic heme by means of the radical
occurs prior to formation of the epoxide.82 Thus, the epoxide is not involved in the CYP inhibition.
The β-oxidation cycle activates Δ4VPA to its Coenzyme
A derivative and, through sequential steps of β-oxidation,
yields the Coenzyme A ester of 3-oxo-2-propyl-4pentenoic acid.83 This final metabolite is believed to be a
Ch33-P374194.indd 690
reactive electrophilic species that alkylates 3-ketoacyl-CoA
thiolase (the terminal enzyme of β-oxidation) by means of
a Michael-type addition through nucleophilic attack at the
olefinic terminus.84 Oxidative stress may also be implicated, at least in part, in valproic acid hepatotoxicity, as
suggested by experimental data on the effect of the drug on
reactive oxygen species.85
E. Troglitazone
Troglitazone
((⫾)-5-[4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-ylmethoxy) benzyl]-2,4-thiazolidinedione) is an
oral insulin sensitizer belonging to the thiazolidinedione
class of compounds used for the treatment of type II
diabetes. Its withdrawal from the US market was the
consequence of the recent occurrence of hepatic failure
leading sometimes to death.
It was first demonstrated that troglitazone is metabolized mainly to sulfate and glucuronide conjugates.86 Also
5/30/2008 6:35:37 PM
IV. Examples of Metabolic Conversions Leading to Toxic Metabolites
691
FIGURE 33.29 Bioactivation of Δ4VPA.
troglitazone is an inducer of CYP3A.87 The mechanism of
toxicity is still unclear, but seems to proceed according to
two distinct pathways. This is supported by the demonstration that incubation of troglitazone with P450 isoforms in
the presence of glutathione give rise to at least five GSH
conjugates.88,89 Identification of these adducts provided
evidence for the two pathways described in Figures 33.30
and 33.31.
As described in Figure 33.30, oxidative cleavage of the
thiazolidinedione ring probably generates highly electrophilic
α-ketoisocyanate and sulfenic acid intermediates. This CYP 3A
mediated oxidation would afford a reactive sulfoxide intermediate, which undergoes a spontaneous ring opening.
The second pathway (Figure 33.31) consists of a CYP3Amediated90 one-electron oxidation of the phenolic hydroxyl
group leading to an unstable hemiacetal, which opens spontaneously to form the quinone metabolite. This undergoes
thiazolidinedione ring oxidation according to the pathway
shown in Figure 33.30. Alternatively, a CYP-mediated
hydrogen abstraction may occur on the phenoxy radical,
leading to an o-quinone methide derivative.
It is now well established that troglitazone undergoes
several metabolic transformation mediated by CYP3A4,
leading to numerous electrophilic species.91 Thus toxicity
Ch33-P374194.indd 691
FIGURE 33.30
Oxidation of the thiazolidinedione ring of troglitazone.
5/30/2008 6:35:38 PM
692
CHAPTER 33 Biotransformations Leading to Toxic Metabolites: Chemical Aspect
FIGURE 33.31 Oxidation of the chromane ring of
troglitazone.
acts probably both by covalent binding to hepatic proteins
and oxidative stress through a redox cycling process. The
implication of the thiazolidinedione moiety is less likely
since the more recent drugs of this series seems devoid of
toxicity. Recent studies using mitochondrial manganese
superoxide dismutase partially deficient mice also suggested
that genetic deficiencies may be, at least partially, responsible for the liver failure in troglitazone-treated patients.92,93
V. CONCLUSION
In the foregoing discussion, it has been emphasized that
almost all metabolic reactions are capable of producing
Ch33-P374194.indd 692
reactive metabolites. This bioactivation yields toxic compounds that may act directly or indirectly56 (Figure 33.32).
The emergence of toxicity may be the outcome of the
interactions of metabolites or reactive intermediates with
biological targets such as cellular macromolecules. Some
compounds exhibit their toxicity by inducing the generation of reactive oxygen species, thus producing alterations
in the redox state of the cell. Often, covalent bonds are
formed during a phenomenon that may be referred to as
“alkylating stress.” Bioactivation of drugs followed by drug
protein adduction is then considered as a key sequence in
the occurrence of toxic side effects.94 As the precise damages of adducts on cellular functions are not fully understood, the formation of electrophilic metabolites is to be
5/30/2008 6:35:39 PM
693
V. Conclusion
TABLE 33.1 Some Major Toxophoric Groups and
Their Bioactivation Mechanisms
Toxophoric group
Bioactivation mechanism
Azocompounds
Acetamides
Aromatic/heterocyclic
amines
Nitrenium ions, tautomeric
carbonium ions
Nitro compounds
FIGURE 33.32
Alkylating and oxidative stresses.
avoided in drug design. Proteome profiling (proteomics)
may help to identify and compare proteins implicated in
alkylating stress due to drugs, but this field remains to be
developed and methods are to be validated. The specific
inhibition of an enzyme by its own substrate is a peculiar
feature of alkylating stress. Determination and monitoring of drug protein adducts have important implications in
drug development, for example, in identifying CYP3A4
inactivation, since this CYP isoform is responsible for the
metabolism of about 50% of the therapeutic drugs.95 Thus
medicinal chemists have set a threshold of acceptable
covalent binding when developing a new drug. For example, this value for covalent binding levels to liver proteins
was less than 50 pmol-equiv/mg protein under standard
conditions at Merck96 and can be subject to discussion on
a case-to-case basis. This target represents about 1/20th of
the level of binding for model hepatotoxins. Often the molecule can be modified to decrease this type of unwanted
reaction without losing too much pharmacological
activity.96–98
Such a variety of mechanisms makes it difficult to
point at molecular functions susceptible to produce toxic
effects through bioactivation. However, some major toxophoric groups may be highlighted (Table 33.1). They may
be implicated in acute or chronic toxicity. These patterns
must be of particular concern in drug design. A number of
recent papers on these matters have been published on how
to avoid those toxic events in drug design.99–101
Ch33-P374194.indd 693
Nitroaromatic
compounds
Radical formation/oxidative stress
Bromoarenes
Arene oxide formation
Ethinyl
Ketene formation/heme
destruction
Furanes
Furane epoxide and ene-dial
formation
Pyrroles
Pyrrole oxide
Nitrogen mustard
Aziridium ions
Nitroso compounds
Hydrazines
Diazonium ions/heme adduct/
radical formation
Nitrosamines
Carbenium ions/DNA alkylation
Polyhalogenated
compounds
Radical and carbene formation/
episulfonium with GSH
Quinone
Semiquinone radical formation/
oxidative stress/thiol trapping
Thioamides
Thiourea formation
Thiophene
Thiophene sulfoxide or thiophene
epoxide formation
Vinyl
Epoxidation/heme destruction
Generally, the formation of toxic metabolites is not the
only pathway of biotransformation, and the overall metabolism is constituted toward detoxication and bioactivation
processes. The toxic metabolites are themselves often further detoxified. The duality between a beneficial detoxication phenomenon (metabolism, drug resistance) and the
occurrence of a toxic effect represents the cost for adaptability of metabolic enzymes to the diversity of xenobiotics. For those interested, a recent review applies the above
chemistry to predict drug safety.102
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