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Table 3.4
Frequency of slow acetylators of different ethnic groups
Population
Frequency of slow acetylators, %
Eskimos of Canada
0.05
Eskimos of Southern Alaska
0.18
Eskimos of Northern Alaska
0.27
Japanese
0.11
The Ainu
0.13
Koreans
0.10
Chinese
0.15
Indians
0.58
Black Americans
0.51
Sudanese
0.65
Amhara (Ethiopia)
0.83
San people
0.30
Egyptian Arabs
0.83
White Americans
0.58
Germans
0.44
American Scandinavians
0.67
American Italians
0.64
Finns
0.64
Sami
0.28
Norwegians
0.52
Russians
0.52
Table 3.5 shows the effect of acetylation phenotype and G6PD deficiency on the relative concentration of hemoglobin adduct of aniline in
the blood of chemical industry workers, who had been exposed to aniline.
The highest relative concentration of the adduct-toxicant is observed in slow
acetylators with G6PD deficiency.
Table 3.5
Effect of acetylation phenotype and G6PD deficiency on relative
concentration of hemoglobin adduct of aniline in blood
Acetylation phenotype
G6PD deficiency
Relative concentration of hemo-
globin adduct of aniline
Fast
Slow
Negative
Positive
+ +
2 +
+
30
+ +
20
+
+
100
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In this instance, G6PD deficiency leads to a decrease in glutathioneS-transferase conjugation (a detoxification process) in Phase II of the biotransformation of aniline. The slow reaction rate of N-acetylation also causes
a decrease in the detoxification of reactive N-hydroxy aniline derivatives.
These reactive aniline metabolic intermediates then accumulate and covalently bind to hemoglobin, causing structural and functional changes in hemoglobin, which further leads to hemolytic anemia. Thus, individual genetic
differences significantly influence the risk of developing an adverse health
effect from exposure to chemical pollutants.
The phenomenon of fast acetylators being slow oxidants and vice
versa is an inherent property for protection against ecotoxicants. This is
a form of inter-process compensation between the biochemical processes
of acetylation and oxidation. The most common pathways of metabolism
and elimination of xenobiotics from the human body involve oxidative processes, which are characteristic of phenotypical variability due to genetic
polymorphisms of the human liver monooxygenase system. For instance,
the oxidation of debrisoquine, an antihypertensive drug, is characteristic
of a bimodal distribution of slow and fast oxidants. Thus, personalized
daily doses of debrisoquine vary significantly from 20 mg for slow oxi-
dants to 400 mg for fast oxidants. The elimination of debrisoquine is partly
by urinary excretion of the drug untransformed. Other pathways of debrisoquine metabolism in humans include aromatic hydroxylation and heterocyclic ring-cleavage.
The role of analytical methods becomes particularly important, given
the potentially massive nature of research, that is required for personalized
clarification of toxicokinetic processes of biotransformation. The data
from such research can be used not only for the optimization of treatment,
but also for professional selection, risk assessment and prevention of injury
to industrial personnel, working with carcinogens and other toxic compounds.
The assessment of monooxegenase activity is carried out with testdrugs, such as antipyrine, isoniazid, butazolidine, barbamil, diazepam, amidopyrine, hexobarbital, quinine, theophylline, and warfarin. The half-life
ranges of some of these drugs, that undergo metabolic oxidation, are presented in Table 3.6.
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Table 3.6
Half-life (t½) of drugs under oxidative metabolism
Drug substance
Half-life (t½) range, hours
Antipyrine
5–22
Butazolidine (phenylbutazone)
30–175
Warfarin
15–70
Diazepam
9–53
Dicoumarin
7–74
Diphenin (phenytoin)
10–42
Indomethacin (Methindol)
4–12
Carbamazepine
18–55
Nortriptyline
15–90
Pyridinol carbamate
3–22
Tolbutamide
3–19
Nevertheless, some of these drugs, including warfarin, quinine, and bu-
tazolidine cannot accurately show monooxegenase activity due to their sig-
nificant binding to plasma proteins, which influences the metabolic path of
the drug.
The antipyrine test is the most reliable and widely accepted method of
oxidation phenotyping, by which a trimodal distribution of phenotypes (fast,
intermediate, and slow metabolizers) had been identified. The average halflife values of antipyrine in phenotypic groups of healthy persons with different rates of oxidative metabolism of the drug are given in Table. 3.7.
Table 3.7
Mean values of the half-life (t
1/2
) of antipyrine in phenotypic groups
of healthy persons (n = 106) with different rates of oxidative
metabolism of the drug
Oxidation
phenotype
Half-life,
hours
Boundary (t½)
Number
Percentage (%)
of total
Fast
8.0
< 10.2
28
26.4
Intermediate
11.6
10.2–13.5
49
46.2
Slow
15.0
>13.5
29
27.4
The first phase of antipyrine metabolism involves its hydroxylation to
form 3-hydroxy-methylantipyrine, norantipyrine, 4-hydroxyantipyrine, 4´-hy-
droxyantipyrine, carboxyantipyrine, and 4,4 -dihydroxyantipyrine (Fig. 3.17).
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Fig. 3.17. Metabolites formed by antipyrine hydroxylation:
I – antipyrine, II – 3-hydroxymethylantipyrine, III – norantipyrine,
IV – 4-hydroxyantipyrine, V – 4´-hydroxyantipyrine,
VI – carboxyantipyrine, VII – 4,4 -dihydroxyantipyrine
The diversity of these metabolites is due to the participation of various
isoforms of cytochrome P450 in the oxidation of antipyrine. The Phase II
biotransformation of antipyrine involves the conversion of the metabolites
into their corresponding conjugates that easily get excreted in urine.
3.6 . I n d i v i d u a l t o x i c o l o g i c a l r e a c t i o n s
Individual sensitivity results into either acute, intermediary, or low ecotoxic effects in different persons with the same exposure level to a xenobiotic. This individual difference can be due to: an elevation or decrease in
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the rate of toxicokinetic processes (biotransformation and excretion), responsible for the removal of xenobiotics from the body; changes in the sensitivity
of individual or systemic receptors to the action of a chemical substance;
the absence of a normal or the presence of an atypical enzyme, responsible
for the biotransformation of a xenobiotic; and genetic pathology of receptors.
The first two factors contribute mainly to quantitative variations in individual
biological activity, while the last two lead to a qualitatively new, usually,
pathological response to a toxicant.
Elimination of xenobiotics from the body is often by two means; i.e. excretion of untransformed xenobiotic and excretion of metabolite of the xenobiotic (Fig. 3.18). These two forms are often first order processes with
the rate of excretion and metabolism dependent on the concentration of untransformed xenobiotic in the body. The elimination rate of a xenobiotic from
the body is determined by both the urinary excretion rate constant (ke) and
the metabolism rate constant (km):
,
where Xb is the concentration of untransformed xenobiotic in the body; dXb
is the change in concentration of the untransformed xenobiotic in the body;
and dt is the duration of the elimination process. Thus, the final elimination
rate constant (kel) is the sum of the urinary excretion rate constant (ke) and
the metabolism rate constant (km): kel = ke + km .
Fig. 3.18. Elimination of xenobiotics from the body
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The rate of biotransformation significantly influences inter-individual
differences in the rate of elimination of many xenobiotics that are largely
excreted in the form of their metabolites. This further results in the differences of therapeutic effectiveness of a drug or the toxic effect of a toxicant
in different persons. Genetically-determined variability in the sensitivity or
resistance to exogenous or environmental factors is dealt with as ecogenetics.
Similarly, such genetically-determined response to drugs is studied as phar-
macogenetics. In this regard, individual phenotypic development is influenced by predisposition genes, which are categorized as metabolic genes,
trigger genes (signal transduction or signal transfer genes) and cell receptor
genes, depending on their role in biochemical processes.
Detection of biochemical changes in endogenous physiological processes, as well as the metabolism of exogenous compounds is very significant in the study of genetic polymorphism. Some enzyme systems under genetic regulation with polymorphisms of biotransformation are presented in
Table 3.8. Some genetic studies have indicated significant differences in allelic polymorphism for inter-population and inter-ethnic groups, reflecting
the peculiarity of living conditions, nutrition and lifestyle of populations in
different regions of the world.
Table 3.8
Enzymes under genetic regulation with polymorphisms
of biotransformation
Enzyme system
Metabolism
Occurrence
Xenobiotic
Low activity of hepatic monooxygenase system
Oxidation
5–30 % (different
nationalities)
Various compounds of different structures
Low
N-acetyltransferase
activity
Acetylation
50 % in Caucasians,
various ratios in
other populations
Amines, hydrazines, hydrazides,
amides, indoles
Low arylesterase
activity
Hydrolysis of esters
50 % in Caucasians
Drugs with the
ester functional
group
Low catalase
activity
Peroxide
decomposition
1 % in Japanese
Peroxides
Low activity
glucuronyltransfer
ase
Glucuronide
formation
Rare
Antibiotics,
barbiturates,
opiates,
sulfonamides
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Mutant isoforms of enzymes have their function impaired in comparison
with their normal forms. Functionally defective genes, resulting in defective
enzymes, are much more common in individuals with various diseases, in
the etiology of which adverse exogenous factors play an important role.
These defective genes can be referred to as predisposition genes of
the diseases they are attributed to (Table 3.9).
Table 3.9
Predisposition genes and their attributed diseases
Gene
Primary defect
Population
frequency, %
Abnormality
GSTM1
Phase II detoxification
disorder
40
Lung cancer, chronic
bronchitis, endometriosis
NAT-2
Phase II detoxification
disorder
50
Breast Cancer
mEPHX
Phase I detoxification
disorder
6
Chronic exacerbations of pneumonia,
emphysema, asthma
CYP1A1
Phase I detoxification
disorder
27
Lung cancer
MTHFR
Homocysteinemia
5
Atherosclerosis
ACE
Elevation of enzyme
activity
30
Myocardial infarction
ApoE
Hyperlipoproteinemia
15
Atherosclerosis
CC16
Secretory protein
CC16 dysfunction
10
Asthma
CCR-5
Loss of lymphocyte
receptor
26
AIDS resistance
For instance, almost 40 % of the Russian population has a defective
(null) allele of glutathione-S-transferase. This genotype is particularly characteristic of persons with chronic obstructive bronchitis, as well as pulmonary and bladder cancers. Individuals with this genotype are more likely to
develop liver cirrhosis against the background of alcoholism. Homozygous
individuals for the recessive allele of the GSTP1 gene are highly susceptible
to developing various tumors, skin cancer and Parkinson’s disease. This severe neurodegenerative disease, arising from selective death of dopaminergic
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neurons in the subcortical regions of the brain, is often seen in persons with
history of chronic exposure to pesticides.
The NAT-2 gene, responsible for the synthesis of the enzyme N-acetyltransferase-2, may contribute to the onset of breast cancer. This effect, however, is directly related to smoking in postmenopausal women. Indeed,
the risk of breast cancer increases by approximately 20 times in postmenopausal women, who are genotypically homozygous for the slow allele of
the NAT-2 gene, and are thus, phenotypically slow acetylators, and had been
smokers in their young ages.
This phenomenon is, however, not observed in phenotypic fast acetylators. Thus, some pathological reactions can be attributed to specific genetic
anomalies with inducive exogenous or environmental factors, including xenobiotics. Examples of ecogenetic pathological reactions to environmental
factors are given in Table. 3.10.
Molecular genetic methods (MGM) are used for the determination of
individual genotypes. However, gene action is a complex process; so the determination of apparent absence of a mutation by molecular-genetic methods
is not always completely conclusive of a normal biochemical phenotype. Molecular genetic methods involve the isolation, as well as fluorescent or radioactive labeling of genes for determining similar DNA fragments in an individual under study. The labeled fragment (gene) of interest is referred to as
a DNA probe (or genetic marker).
DNA isolation can be from an organ, tissue, blood cells or cells cultured
in vitro. The isolated fragments can be broken down by nucleases to obtain
DNA fragments of different lengths. Molecular genetic methods are complemented by analytical biochemical methods that determine phenotype, which
ultimately determines disease conditions and toxic effects of xenobiotics in
an organism. In fact, biochemical parameters, such as primary proteins synthesized from genes, as well as metabolites of exogenous substances in cells
and extracellular biological fluids, more adequately indicate disorders that
can lead to pathological conditions. This is due to the fact that, analytical
methods aimed at detecting genetically-determined biochemical parameters
indicate biochemical phenotype. The diversity of analytical technologies
used in phenotypic studies is related to the various levels of phenotype determination; i.e. from gene products (proteins) to final metabolites of a testdrug. Thus, these analytical methods have become very relevant in the diagnosis of hereditary disorders, as well as the assessment of risk of toxicity and
carcinogenesis. Accidental or emergency situations may result in acute toxic
effects of both large and small concentrations of physiologically active
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compounds in the environment. Genes, encoding enzymes that are responsible for the metabolism of chemical compounds, can influence toxicity and
carcinogenesis caused by the combined effect of biological, chemical and
physical factors. There are, however, genes that get expressed only under
the influence of specific environmental factors, in the absence of which these
genes remain inactive. These are called silent genes.
Table 3.10
Ecogenetic pathological responses to environmental factors
Environmental
factor
Inducive factor
Affected enzyme
Pathological reaction
Climatic
conditions
Cold weather
Α1proteinase
inhibitor
Increased risk of colds
Solar radiation
DNA Repair
Enzymes
Skin ulcerations,
cancer
Industrial
environment
Dust
Α1proteinase
inhibitor
Obstructive pulmonary
disease
Hypoxia,
Nitrofurans
Glucose-6phosphate
dehydrogenase
Hemolysis of
erythrocytes
Benzidine dyes
Acetyltransferase
Bladder cancer
Photoactive
substances
Transferin
Photodermatosis
Household
Hazard
Smoking
Α1proteinase
inhibitor
Obstructive pulmonary
disease
Alcohol
Aldehyde
dehydrogenase
Pathological sensitivity to alcohol
Food products
Milk
Lactose
Lactase
Lactose intolerance,
dyspepsia
Galactose
Galactose-1phosphate uridyltransferase
Galactosemia
Vitamin C
deficiency
L-gulonolactone
oxidase
Scurvy
Horse beans
Glucose-6phosphate
dehydrogenase
Favism
Fructose, Sucrose
Aldolase
Hereditary fructose
intolerance
Nevertheless, modern molecular genetic methods have the capacity of
identification of these genes, and thus, the individual hosts of them. Potentially toxic environmental factors usually have adverse effects on only genetically predisposed persons but not on an entire population. A greater risk
of toxicity and carcinogenesis, arising from exposure to toxicants, is observed in persons with high activity of Phase I enzymes but low activity of
Phase II enzymes (Fig. 3.19).
Fig. 3.19. Level of risk of toxicity, depending on combinations of level
of toxic exposure and genetic polymorphism of Phase I and II
biotransformation
Indeed, the predictive role of molecular-genetic methods is very relevant in establishing genetic predisposition to the development of carcinogenesis and the adoption of preventive measures.
Q u e s t i o n s & A s s i g n m e n t s
1. What is toxicokinetics?
2. What physicochemical processes occur in the process of distribution
of exogenous substances in the human body?
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