Добавил:
ivanov666
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Fundamentals of Toxicology. Tutorial
.pdf
51
correlates with its coefficient of distribution in a two-phase medium of a nonpolar solvent (e.g. heptane, chloroform) and water. Substances with relatively high to moderate solubility in water are excreted untransformed in
urine, bile and other biological fluids. Most xenobiotics are lipophilic, and
thus, undergo biotransformation to produce water-soluble metabolites, which
are excreted mainly in urine.
The first phase (Phase I) of biotransformation consists of hydrolytic,
oxidative, or reductive transformation, by which the molecule of a xenobiotic
is enriched with polar groups, making it reactive and more soluble in water.
The second phase (Phase II) involves synthetic metabolic processes of conjugation of intermediate products with endogenous molecules, resulting into
the formation of polar compounds, which are excreted from the body by various mechanisms. The chemical modification of a xenobiotic molecule may
result in attenuation of toxicity, increased toxicity, change in the nature of toxic
effect, or initiation of a toxic process.
Fig. 3.9. Phases of biotransformation of drugs
In most cases, metabolism leads to the formation of less active and less
toxic metabolites, a phenomenon which can be said to be a natural defense
mechanism of the body against xenobiotics. Thus, thiocyanates are several
hundred times less toxic than cyanides from which they are formed in the process of biotransformation. Similarly, hydrolytic cleavage of the fluorine ion
from molecules of sarin, soman, and diisopropyl fluorophosphate leads to
loss of these substances’ ability to inhibit the activity of acetylcholinesterase
and a significant decrease in their toxicity. However, the body is incapable of
52
envisaging the entry of chemical substances, the biotransformation of which
results in reactive and toxic metabolites, as well as their accumulation.
The anabolic or catabolic metabolism of chemical compounds into
more toxic forms is referred to as toxification or toxication. However, the bi-
osynthesis (anabolism) of a toxin from a non-toxic precursor is called lethal
synthesis or suicide metabolism. In the metabolic process of N-dealkylation
of narcotic drugs, such as morphine and codeine (Fig. 3.10), the corresponding norderivatives formed are more toxic than the drugs themselves.
Fig 3.10. N-dealkylation of morphine and codeine
Indeed, norcodeine is found to be six times more toxic than codeine and twice
more toxic than morphine under the same conditions of administration.
The drug, cimetidine, used in the treatment of heartburn and peptic ulcers, can
undergo N-nitrozation with nitrites from food in the stomach. Oxidative hydroxylation and cleavage of products of the nitrozation reaction forms carcinogenic nitrosamines that can cause gastric cancer (Fig. 3.11). Thus, biotransformation does not always lead to detoxification of xenobiotics. One of the major
metabolic pathways of toxic substances involves the formation of free radicals
from the substances (Fig. 3.12). The hepatotropic poison, carbon tetrachloride
causes necrosis and fat degeneration of the liver even in doses of 1 μL per 100 g
of body weight. Decomposition of carbon tetrachloride occurs with the formation of free radicals, which damage enzyme systems and have a peroxidative effect that initiates a chain reaction of lipid peroxidation.
53
Fig. 3.11. Scheme of toxification of cimetidine
54
Fig. 3.12. Metabolism of toxicants involving the formation of free radicals
Enzymes that catalyze and regulate biotransformation reactions are located mainly in cells of the liver, where metabolism of xenobiotics occurs.
Xenobiotic metabolism is carried out by functionalization (Phase I) and conjugation (Phase II) reactions. Phase I reactions involve the activation of xenobiotics by enzymes of the cytochrome P450 superfamily, forming shortlived intermediate electrophilic metabolites that may have genotoxic properties. In Phase II, these intermediate metabolites, with the help of enzymes of
the glutathione transferase, UDP-glucuronosyltransferase, and N-acetyltransferase families, are converted into water-soluble non-toxic products and
excreted from the body. The enzymes involved in metabolism of xenobiotics
55
are mainly intracellular; a few of which are in the cytosol and mitochondria,
while most of them are associated with the smooth endoplasmic reticulum
(Table 3.3).
Many transformations of xenobiotics in the human body are carried out
by hepatic microsomes. The microsomal fraction of hepatic homogenate catalyzes both the reduction of azo- and nitro- compounds and the oxidative
demethylation of amino azo compounds. Oxidation reactions, such as hydroxylation of aliphatic, acyclic and aromatic compounds, S- and N-oxidation, deamination, O-, N- and S-dealkylation, and dehalogenation also occur
in microsomes.
Table 3.3
Localization of metabolic reactions of xenobiotics
Reaction
Localization
Phase I
Oxidation:
– hydroxylation
– decarboxylation
– formation of oxides
– desulfurization
– dehalogenation
– oxidation of alcohols
– oxidation of aldehydes
Reduction:
– reduction of aldehydes
– azo reduction
– reduction of nitro compounds
Hydrolysis:
– splitting of esters
– breakdown of amide bonds
Microsomes
Microsomes
Microsomes
Microsomes
Microsomes
Microsomes, Cytosol
Cytosol
Cytosol
Microsomes
Microsomes, Cytosol
Microsomes, Cytosol
Microsomes, Cytosol
Phase II
Conjugation with glucuronic acid
Conjugation with sulfates
Acetylation
Glutathione conjugation
Methylation
Microsomes
Cytosol
Microsomes, Cytosol
Cytosol
Cytosol
56
Hydroxylation reactions of xenobiotic metabolism in the microsomal
system are initiated by the activation of oxygen, which is then transferred to
the substrate (Fig. 3.13). Major enzymes of biotransformation include the fla-
voenzyme NADP·H-cytochrome C reductase, cytochrome P450 and
NADP·H-cytochrome P450 reductase. The participation of cytochrome P450
in drug metabolism is proven by the fact that oxidation of endogenous and
exogenous substrates take place in the liver and adrenal cortex, the microsomes of which are rich in cytochrome P450.
а
b
Fig. 3.13. Hydroxylation of xenobiotics by: microsomal monooxidases (a);
and cytochrome P-450 (b)
Enzymes of the cytochrome P450 system are referred to as microsomal
oxidases (MO), monooxygenases, or microsomal metabolic system. In recent
years, there has been isolation of isoenzymes of the cytochrome P450 superfamily, responsible for the biotransformation of various drugs and ecotoxicants.
The enzymatic conjugation system plays a special role in the metabolism of xenobiotics. Conjugation is the process of binding toxic substances
or their metabolites with groups of endogenous molecules. The products of
these reactions are called conjugates. The physicochemical properties of conjugates differ significantly from the properties of reactants from which they
are formed. The toxic activity of the reactants is either lost or drastically reduced in their corresponding conjugates, which are easily excreted. A common conjugation reaction, involving the addition of a methyl group onto
a substrate, referred to as methylation, can be accompanied by increase or
decrease in biological activity of the substrate. The amino acid, methionine
57
serves as the donor of the methyl group, and thus, converting into homocysteine in the process. The reaction is categorized as N-, O- and S-methylation,
depending on the functional group of the substrate that gets methylated.
For example, phenylethanolamines are methylated on their primary amine
group with the participation of the enzyme, N-methyltransferase in the adrenal medulla. Nevertheless, pulmonary, hepatic, and brain tissues have
the highest concentration of N-methyltransferase. The process of methylation often occurs with primary, secondary and tertiary amines. Common primary amines that undergo methylation are tryptamine and serotonin.
A large group of secondary amines methylated by N-methyltransferase
include normorphine, norcodeine, nornicotine, N-methylserotonin, etc.
The methylation of tertiary amines, such as pyridine and quinoline results in
their conversion into quaternary amines. The O- and S-methyltransferases
catalyze the methylation of substrates; on an oxygen or sulfur atom, respectively, in the molecule of the substrate.
Acetylation, the conjugation of a chemical substrate with acetic acid
residue (Fig. 3.14) is characteristic of the biotransformation of many exogenous and endogenous substances with primary amine groups in their structure. This reaction is catalyzed by arylamine acetyltransferase (AAT), which
is highly coenzyme-specific and less substrate-specific.
Fig. 3.14. N-acetylation of aromatic amines
Xenobiotics that undergo biotransformation by acetylation include primary arylamines, such as sulfonamides, novocaine, novocainamide, derivatives of p-aminophenolic and p-aminobenzoic acid, as well as isonicotinic acid
hydrazide (isoniazid), clonazepam (after reduction of the nitro group), mescaline, aromatic and aliphatic amines, hydrazines, and endogenous substrates,
such as choline, serotonin, histamine, etc. In mammalian tissues, particularly,
xenobiotics that undergo acetylation include, but not limited to isoniazid, hydrazine, benzidine, 4-aminoantipyrine, aniline, sulfadiazine, sulfamerazine,
furfurylamine, serotonin, tryptamine, histamine, α-naphthylamine, and methylene bis-o-chloroaniline. However, acetylation of arylamines sometimes
58
leads to toxication; for instance, the acetylated products of sulfonamides, due
to poor solubility in aqueous medium, crystallize in and affect the function of
the urinary tract. The extent to which acetylation occurs varies with different
xenobiotics. For instance, the total content of acetyl derivative of sulfadiazine
in blood and urine can reach up to 93 % of the administered dose, while the effectiveness of acetylation of etazole and norsulfazole is 17 and 40 %, respectively.
Biotransformation of xenobiotics occurs in various organs and tissues
(lungs, placenta, skin, intestinal mucosa); however, most of the transformations occur in the liver with the participation of enzymes localized in
the endoplasmic reticulum of hepatocytes. Variations in the rate of biotransformation of a xenobiotic in different people lead to individual differences in
the toxicokinetics of the xenobiotic. Such differences, consequently, influence the toxic effect of the xenobiotic in different persons with equal exposure. The rate of biotransformation of xenobiotics depends on genetic deterministic activity of enzymes. In addition to genetic factors, the activity of
enzymes can be significantly influenced by external factors, such as tobacco
smoke, food, alcohol, gender, age, concomitant diseases, etc.
Toxicokinetic studies to determine the rate of biotransformation of xenobiotics are carried out by phenotyping and genotyping. Phenotyping is a di-
rect determination of the activity of a particular metabolic enzyme, based on
the toxicokinetic parameters of a test drug and (or) its metabolite. Genotyping, however, is an indirect method based on the study of polymorphism of
the genes that encode for a metabolic enzyme, using polymerase chain reaction (PCR). The rate of metabolism of a xenobiotic can be estimated based
on the metabolic ratio of the concentration of the xenobiotic to the concentration of its metabolite or derivative in blood plasma, urine, or saliva.
This helps to distinguish between groups of patients with variations in the activity of an enzyme, responsible for the biotransformation of a xenobiotic.
Fast metabolizers are persons with high metabolic rate of a particular xenobiotic. In this group of persons, the concentration of a test-drug after administration, in a biological fluid at a given time, is lower than the concentration
of its metabolite, due to the occurrence of intensive metabolism of the drug.
Thus, in such persons, a larger dose of the drug is required to achieve an
intended therapeutic effect. Intermediate metabolizers are characterized by
“normal” metabolic rate of a test-drug. In this group of persons, there is less
significant difference in the concentrations of a test-drug and its metabolite
in a biological fluid at a given time after administration. Slow metabolizers,
in contrast to fast metabolizers, are characterized by low metabolic rate of
59
a test-drug. In this group of persons, the ratio of the concentration of a testdrug to the concentration of its metabolite is high, since much of the drug
remains untransformed over a longer period. Thus, a smaller dose of the drug
is recommended to be administered to such persons to prevent its accumulation in organs and tissues, leading to drug toxicity. All these groups of metabolizers are characterized by a genetic polymorphism, leading to bimodal
or trimodal distribution of persons in a population (Fig. 3.15, 3.16).
Fig. 3.15. Bimodal distribution: Half-life of isoniazid in fast
and slow metabolizers
Fig. 3.16. Concentration of isoniazid in blood plasma 6 hours after its
administration at a dose of 9.8 mg/kg body weight in 267 volunteers
60
There are two isoenzymes of N-acetyltransferase: N-acetyltransferase
1 (NAT1) and N-acetyltransferase 2 (NAT2). Only a small amount of arylamines are acetylated by NAT1, which is not associated with any genetic polymorphism. Thus, acetylation reactions are mainly catalyzed by NAT2.
The slow phenotype of NAT2 is associated with the development of a number of diseases, as well as toxic reactions, resulting from the accumulation of
drugs in organs and tissues, due to their slow biotransformation. Consequently, the study of NAT2 activity or its modification in pharmacotherapy
is relevant for the prognosis of the safety and adequate administration of
drugs. The assessment of NAT2 activity and the determination of acetylation
phenotypes is very important for the prognosis of side effects, as well as the
selection of individual optimal doses of drugs. The proportion of fast and
slow acetylator phenotypes of a population differs with ethnic or geographical origin of the population. Most European and North American populations consist of 40–70 % slow acetylators, Egyptians – 80 %, while populations along the Pacific Asian coast (e.g. Japan, China, Korea, and Thailand)
have only 10–30 % slow acetylators. A very low number of slow acetylators
(less than 10 %) is observed among the Eskimos (Canada) and inhabitants of
Papua New Guinea. Approximately 43.45 % fast and 56.55 % slow acetylator phenotypes had been observed among inhabitants of Western Russia.
Though, the reason for this phenotypic distribution is not fully understood, it
is assumed that NAT2 activity is significantly influenced by physicochemical
environmental conditions and differences in dietary habits.
The reaction rate of the process of acetylation is a major factor in determining therapeutic effectiveness and toxicity of drugs that undergo the
acetylation metabolic pathway. Indeed, the process of acetylation is one of
the first metabolic pathways for which genetic deterministic distribution into
phenotypic groups has been established. The frequency of distribution of
slow acetylators in various ethnic groups and geographical locations of some
countries is presented in Table 3.4. Nevertheless, available data on population distributions of acetylator phenotypes are conflicting due to the influence
of methodological factors and pathological conditions on the results of phenotypic studies.
The occurrence of more than one toxicogenetic influence may lead to
significant differences in the risk of toxic effects arising from exposure to
toxicants. For instance, persons with glucose-6-phosphate dehydrogenase
(G6PD) deficiency together with slow acetylation phenotype respond adversely to aromatic amines.
Соседние файлы в предмете [НЕСОРТИРОВАННОЕ]