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Файл:Fundamentals of Toxicology. Tutorial
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Xenobiotics (from Greek xenos – alien and bios – life) are compounds,
that are foreign to a living organism or system, and are artificially synthesized; that is, substances that are not formed in a living organism and are not
characteristic of it. Substances formed in an organism are referred to as endogenous, and those formed outside the organism are exogenous (foreign to
a living organism).
Tolerance of an organism or ecosystem is its ability to continually subject to a certain amount of a chemical substance without developing toxic
effects. The toxic effect of a chemical substance, which is based on its interaction with a biological system at a molecular level, leads to impairment of
the functions of that biological system. Thus, the chemism of the interaction
between a toxicant and a biological system determines the mechanism of
toxic activity of the toxicant. The pathways of toxicity of a chemical substance is a consequence of the substance’s toxic activity.
The structural component of a biological system with which a toxicant
chemically interacts is called a receptor or target. Receptors are molecules
on bio-substrates for specific binding to xenobiotics (or endogenous molecules), by which the binding process is in accordance with the law of mass
action. Molecules of proteins, nucleic acids, polysaccharides or lipids can act
as receptors. The fragments of biomolecules that directly form complexes
with toxicants are called binding sites. For example, the hemoglobin molecule is a carbon monoxide receptor, the binding site of which is the ferrous
(Fe2+) ion, enclosed in the porphyrin ring of heme.
The evolutionary process of organisms is accompanied by the formation
of special molecular complexes, such as enzymes, hormones, immunoglobulins, neurotransmitters, etc., that act as bioregulators, and are components of
biological systems with high affinity for different chemical substances. Selec-
tive receptors are parts of biological systems that have high affinity for specific chemical substances. Indeed, in many cases, the receptors are enzymes.
For instance, the enzyme, acetylcholinesterase (AchE) is a receptor with
the hydroxyl group of serine in its active site, serving as the binding site for
organophosphorus substances, such as diazinon, ethion, fenthion, malathion,
parathion, etc. This results in the development of specific anti-cholinesterase
effect, caused by organophosphorus compounds. Other binding sites for substances of primary toxicity include amino acid residues, such as histidine and
cysteine in the active site of enzymes, as well as nucleotide and nucleic acid
residues. Binding sites of receptors are usually reactive sulfohydryl, hydroxyl,
carboxylic, amino- or phospho- functional groups, which play vital roles in
cell metabolism.
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Any chemical substance, in order to produce a biological effect, must
have two independent properties: affinity for a receptor and a physicochemi-
cal activity. The simplest idea of toxicity in the light of receptor theory is
given by Clark’s Occupancy Theory: the toxic effect of a substance is propor-
tional to the area of receptors occupied by the molecules of the substance.
The maximum toxic effect of a substance is realized, when a minimum number of its molecules are able to bind to and disable or stimulate vital target
cells. For example, paralysis can be caused by eight molecules of toxins of
Clostridium botulinum, accumulating in a peripheral motor nerve cell ending.
Thus, 1 mg of this substance can be hazardous to 1200 tons of a living organism; and 200 g of it is capable of destroying the entire human population on
earth. The importance of the rate of formation of receptor-toxicant complexes,
their stability and ability for reverse dissociation is often much more than
the degree of saturation of receptors with the toxicant. Structural similarity between a toxicant and a particular metabolite, mediator, hormone, etc., results
into high specificity of the interaction of the toxicant with a target cell.
In this sense, the interaction of a toxicant with a receptor can be likened to
them being fit for each other like a “key and lock”. However, the toxic effect
of many substances has no strict selectivity. Their interference in life processes is based not on specific chemical effects with certain cellular receptors, but on the interaction with the entire cell as a whole. This principle
probably underlies the narcotic mode of action of a variety of organic and
inorganic substances, having the similar property of being non-electrolytes.
The structure and properties of peptide receptors are encoded by specific
genes. These receptors can be silent or active. A silent receptor is a structural
component of a biological system, the interaction of which with a substance
does not lead to a response. An example is the human serum albumin,
the binding of which to xenobiotics or endogenous substances produces no
physiological response. Toxic responses are produced when toxicants interact
with active receptors. The greater the number of active receptors interacting
with a toxicant, the more the impact of toxic effect of the substance. The toxicity of a substance is inversely proportional to its amount, that binds to silent
receptors. The higher the toxicity of a substance, the more effectively it interacts with active receptors; and the importance of an affected biological system,
as well as its receptors for maintaining homeostasis of an organism is measured by the toxicity of substances that they interact with.
Any cell, tissue or organ contains a significant number of receptors, that
can interact with toxicants, triggering various biological reactions. The binding of a xenobiotic to a receptor is a selective process, that occurs within
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a particular concentration range. An increase in concentration of a xenobiotic
in a biosystem leads to an increase in the number of receptor types with which
the xenobiotic interacts; and consequently, a change in its physiological activity. Targets for toxic effects can be intercellular structural elements, cells
and cellular activity regulatory systems.
The toxicity of a chemical substance arises, and can only be studied in
the process of its interaction with a biological system, such as a cell, tissue,
organ, an organism or a population. Mechanisms of toxic action of a chemical substance, as well as their qualitative and quantitative characteristics are
determined by the structure of the substance and its dose in a biological system. Furthermore, the toxic effect of a chemical substance is dependent on
the type and properties of the biological system that the substance is acting
on. In other words, the impact of toxicity of a chemical substance is determined by the level of organization (cell, tissue, organ, organism, population)
of the biological system affected by the substance. In this regard, the main
form of toxic effect of chemical substances is intoxication (poisoning).
The study of the toxic effect of a chemical substance at the cellular level
is an assessment of cytotoxicity of the substance, involving direct effect of
the substance on structural elements of the cell. Toxic effects of chemical
substances at the cellular level include:
– reversible structural and functional changes in the cell; including
changes in shape, mobility, interaction with other chemical substances, etc.;
– premature cell death through necrosis; and
– mutations due to genotoxicity of the chemical substances.
The study of the toxic effect of a chemical substance on individual organs and organ systems involves the assessment of its cardiotoxicity (impact
on heart function), hematotoxicity (effect on blood), hepatotoxicity (effect on
liver function), nephrotoxicity (damage to kidney function), neurotoxicity
(damage to neurons and functions of the nervous system), pulmonary toxicity
(damage to the lungs), etc. The phenomenon of toxicity of chemical substances on organs or organ systems is evident in a variety of organ dysfunctions, such as hyper- or hypotension, increased breathing rate, frequent urination, increased rate of leukopoiesis, etc. Toxic effect at the level of a whole
organism results into poisoning and changes in tolerance to the effects of
physical, chemical and biological factors of the environment, mental and
physical stress, allergies, immunodeficiency, fatigue, etc. At the population
level, ecotoxic effect of chemical substances can be explicit in the form of:
– increased morbidity, mortality, birth defects, as well as decreased fertility;
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– disruption of demographic parameters, such as age and gender ratios
of the population; and
– decreased average life expectancy of an individual in the population.
1 . 2 . C h a r a c t e r i s t i c s o f t o x i c i t y
Toxicity of a xenobiotic is revealed by its interaction with a biological
system; and its magnitude depends on the properties of the xenobiotic and
the biosystem itself. In this regard, the ability of a xenobiotic to reach a biological structure, the interaction with which initiates a toxic process, plays an
important role. Similarly, the type and strength of bond formed between
the xenobiotic and the biostructure, as well as the function of the biostructure
in maintaining homeostasis of an organism, are equally important for the determination of toxicity of the xenobiotic. The structure of a substance determines its molecular size, mass, solubility, volatility, state of aggregation and
reactivity.
The molecular size of a toxicant affects its biological activity; such that,
increase in molecular weight hinders the process of absorption and distribution of the toxicant in the tissues and organs of an organism. Low molecular
weight, chemically inert substances in the form of a gas or solution easily
penetrate into the blood through the lungs, gastrointestinal tract or skin, and
are quickly distributed in tissues, passing through various biological barriers.
The ability of low molecular weight compounds to penetrate barriers is
largely determined by their solubility. Hydrophilic molecules, even with
a molecular weight of 50 to 100 Da, have a limited ability to penetrate mucous membranes. Similarly, the process of penetration of biological barriers
is very much inhibited for polymeric compounds. On the other hand, fat-soluble substances, despite their large molecular size, pass through biological
barriers relatively easily. High molecular weight compounds, with poor solubility in water and lipids, do not penetrate into the internal environment of
organisms; and thus, generally, do not have any toxic effect.
The number of possible isomeric forms of a toxicant, as well as the
selectivity of its toxic action increases with increasing molecular weight.
The biological activity of a substance significantly depends on its molecular
conformation; that is, a spatial configuration of the atoms in its molecule,
that enables it to interact with receptors. The larger the molecule, the more
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evident this dependence. On the contrary, compounds with small molecular
size have a limited number of isomeric forms; and therefore, have an increased number of sites of nonspecific binding in an organism. The number
of toxicants with equal molecular weight, similar chemical structure, but varying toxicity significantly increases with increasing molecular size. For instance, there are over 100 isomers of tetrachlorodibenzo-p-dioxins, among
which the most toxic is 2,3,7,8-tetrachlorodibenzo-p-dioxin.
The probability of a toxicant interacting by van der Waals forces with
a bio-substrate increases with increasing molecular size of the toxicant.
The larger the size of the molecule, the greater the number of atoms of
the toxicant that have contact with the binding site of a receptor; and thus,
the stronger the bond formed. Bioregulators, such as neurotransmitters and
hormones interact with cellular, tissue and organ receptors by weak van der
Waals forces, which break after some time, resulting in the dissociation of
bioregulator-receptor complexes. A toxicant can interact with receptors by
mimicking its biologically active endogenous analogue, that has similar
chemical structure to it. Such mechanism of mimicry underlies the toxic effect of cardiac glycosides and alkaloids, such as caffeine, morphine and nicotine. A toxicant with a significantly larger molecular size than its endogenous substrate binds more firmly to its receptor due to stronger van der Waals
forces that arise between them.
The molecules of toxic chemical substances can be flexible or static,
having a fixed spatial configuration. For instance, alkaloids, polyhalogenated
dibenzofurans, and benzo(a)pyrene are composed of bridged cyclic radicals
that are often held in rigid conformation with very little flexibility. Molecules
that have aliphatic chains or groups in their structure always have an indefinite conformation due to continuous change of the interposition of functional
groups at different times. These substances, however, reach the most stable
spatial conformation at a point in time.
A large number of highly toxic chemical substances exist in isomeric
forms. In low molecular weight substances, such as dichloroethane, differences in the spatial configuration of isomers have little effect on their biological activity. These substances cause low- or non-selective effects; including
the disruption of the permeability of biological membranes and the formation
of covalent bonds with biomolecules, such as lipids, nucleic acids and proteins. Isomeric forms of toxic chemical substances with large molecular size
show significant differences in their toxic effects, interacting mainly with spatially organized highly selective receptors of endogenous bioregulators.
The molecular mass of known neurotransmitters lies in the range of 160
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to 190 Da. For example, acetylcholine and adrenaline molecules consist of
26 atoms, serotonin – 25, and norepinephrine – 23. Obviously, toxicants that
interact with the receptors of these neurotransmitters have similar molecular
sizes, as well as specific spatial configuration; demonstrating the role of isomerism in the biological activity of chemical substances. This principle applies
to competitive inhibitors of many enzymes; such as in the inhibition of acetylcholinesterase by organophosphorus compounds and carbamates. The effect
of isomerism on the toxicity of substances is manifested as follows:
– the more specific the interaction of a substance with a biostructure,
the more distinct the differences in the action of its isomers;
– the higher the toxicity of a substance, the more significant the differences in the biological activity of its isomers;
– if the asymmetric atom in a toxicant molecule occupies an important
position that determines its toxic effect, then the differences in toxic effect of
its isomers are significant. On the contrary, if the asymmetric atom is in a position that does not determine biological effect, then the stereoisomers have
almost the same level of toxicity;
– the more spatially rigid the conformation of a biostructure, the more
pronounced the differences in the activity of the isomers of the toxicant interacting with it.
Physicochemical properties of chemical substances have a determining
effect on their toxicity. These include solubility in water and lipids, as well
as acidity and alkalinity. The solubility of a substance varies in different solvents. Water solubility of a toxicant is a necessary condition for its penetration into the internal aqueous environment of an organism. Substances that
exhibit either properties of acids or bases can be found in protonated or
deprotonated forms in solution; having varying degrees of solubility and toxicity. Fat solubility is also important for processes of biomembrane penetration and distribution of large molecules of toxicants in an organism. The degree of fat solubility of a substance is inversely proportional to the degree of
elimination of the substance from an organism. In other words, the more fatsoluble a substance is, the lesser the substance is easily excreted from
the body. Fat solubility of a toxicant is a measure of the amount of the substance that dissolves in a unit volume of oil or organic solvent. Substances
that are insoluble in fats can enter the body from the environment through
the pores of biological membranes, or by other transport mechanisms across
biological barriers. Among substances of low toxicity are fat-insoluble toxicants with large molecular size. Fat solubility and water solubility are relative
properties of chemical substances. Polar molecules are highly soluble in water,
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but have poor solubility in lipids. As a rule, toxicants with intermediate solubility have the highest biological activity. For some substances, biological activity is proportional to the value of their partition coefficient; that is, the ratio
of their solubility in oil to their solubility in water (solubility
(in oil)
: solubil-
ity
(in water)
) or the ratio of their solubility in heptanes to their solubility in water
(solubility
(in heptanes)
: solubility
(in water)
). Table 1.2 shows increasing toxicity of
homologous series of aliphatic alcohols with increasing molecular weight, decreasing solubility in water, and thus, increasing solubility in heptanes.
Table 1.2
Toxicity of homologous series of aliphatic alcohols
Alcohol
СН3ОН
С2Н5ОН
С3Н7ОН
С4Н9ОН
С5Н11ОН
С6Н13ОН
С8Н17ОН
LD50, g/kg
5.6-7
9
2.2–5.4
0.8–3.7
0.37–4.5
0.71
0.53
The acidity or alkalinity of a toxicant (a weak acid or weak base) determines the ratio of its protonated or deprotonated form to its molecular form
in solution. The ionization of a toxicant is often accompanied by an increase
in its affinity for a receptor, which may occur simultaneously with decreasing
ability to pass through biological barriers. Strong acids and bases completely
dissociate in aqueous solutions, drastically altering the pH of the solution.
Acting on the tissues of an organism, these strong acids and bases cause denaturation of cellular macromolecules; a process that underlies chemical
damage to integumentary tissues.
The interaction of a toxicant with biostructures of an organism conforms with the principles of in vivo chemical reactions and, therefore, largely
depends on its chemical properties. However, most highly toxic compounds
are chemically inert molecules. Intermolecular interactions between toxicants and biomolecules result in the formation of complexes that can easily
undergo dissociation reactions, leading to the restoration of initial properties
of bioreceptors. Based on this principle, the toxic action of a chemical substance can be terminated by removing the unbound amount of the substance
from the body to shift chemical equilibrium towards dissociation of the toxicant-biocomponent complex. If the bonds formed between the toxicant and
a biostructure are strong, the toxicant-biotarget complex can be broken with
chemical reagents that form more stable complexes with the toxicant. For instance, the restoration of acetylcholinesterase activity, inhibited by organophosphorus compounds, is achieved with oximes, that strongly interact with
the toxicants, causing dephosphorylation of the active site of the enzyme.
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The interaction of toxicants with biological targets results in the formation of various types of chemical bonds. Toxic effect due to ionic interac-
tions (binding energy 20 kJ/mol) results from the formation of a water-insoluble complex of a toxicant-ion with a biologically significant biostructureion. For example, water-insoluble calcium fluoride is formed from the interaction between fluoride ion and calcium ion in the case of fluoride poisoning.
This leads to calcium deficiency in the organism. Similar toxic effect occurs
in the case of ethylene glycol poisoning, where the dihydric alcohol undergoes oxidization to form oxalic acid, which then forms a complex with calcium ion (Fig. 1.2).
Fig 1.2. Toxic effect of ethylene glycol
The amount of toxicant-target complexes formed in an organism depends on the pH of its biological fluids, since the degree of dissociation of
many substances in aqueous solution depends on the pH of the medium.
The high stability of interaction in the case of covalent bonds (binding
energy 70 to 600 kJ/mol) makes the binding of toxicant to a biostructure almost irreversible. For instance, organophosphorus compounds, as well as
other common toxicants, such as methyl bromide, methyl chloride, ethylene
oxide, etc., form covalent bonds with the amino acid, serine in the active site
of acetylcholinesterase, inhibiting the enzyme’s activity. As mentioned ear-
lier, due to the strength of bonds formed, the toxicant-biostructure complex
can only be broken with chemical reagents that form more stable complexes
with the toxicant.
In donor-acceptor interactions (binding energy 5 to 20 kJ/mol), metal
cations, such as Zn2+, Cu2+, Fe2+, which are co-factors of many enzymes, often act as acceptors. For example, carbon monoxide interacts with Fe2+ in
hemoglobin, resulting in the formation of carboxyhemoglobin and consequently, deficiency in oxygen transport.
Hydrogen bonds (binding energy 5 to 30 kJ/mol) are very important for
maintaining the spatial structure of proteins, nucleic acids and other
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biomolecules. Substances that are capable of breaking hydrogen bonds can
disrupt the spatial structure of these macromolecules, rendering them nonfunctional.
The energy of van der Waals forces (1 to 5 kDa/mol) between a toxicant, such as aromatic or heterocyclic radicals, and non-polar side chains of
amino acids increases significantly with increasing number of contact sites
between the toxicant and biostructure molecules. In the formation of toxicant-biotarget complexes, van der Waals forces can provide a very strong
fixation of the xenobiotic due to very high binding energy of close interacting
large non-polar molecules. The acting force responsible for bond formation
is inversely proportional to the seventh power of the distance between the interacting molecules; thus, it cannot ensure the attraction of freely-circulating
toxicant molecules in biomedia to a bioreceptor. In this instance, forces of
electrostatic interaction play a paramount role in the process of bringing toxicant molecules and a bioreceptor into contact; after which van der Waals
forces ensure the spatial configuration of the toxicant-bioreceptor complex.
The presence of different functional groups of varied polarities in
chemical substances accounts for the various types of bonds formed in
the process of interactions between chemical substances and biotargets.
The attraction of molecules of chemical substances to bioreceptors is often
by ionic interactions. Specific types of bonds are formed between molecules
of a toxicant and the polar or non-polar groups in the structure of a biotarget.
The more complementary a toxicant molecule is to a bioreceptor, the more
stable the resulting toxicant-receptor complex formed.
1 . 3 . T o x i c i t y o f n a n o m o l e c u l e s
The production and study of the properties of nanomaterials have attracted much attention in recent times. Despite the potential of their wide
practical applications, there are fears of some of them being unknowingly
toxic. The size of nanomolecules contributes to the characteristics of their
interaction with living systems, and thus, their impact on the human organism. Toxicity of a chemical substance with a nanometer-sized structure can
differ significantly from the toxicity of a macroscopic particle with the same
chemical composition. The potential risks associated with the synthesis and
applications of nanomaterials are relevant topics of scientific discussion.
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In this regard, nanotoxicology emerged as the science of hazardous effects of
nanomaterials on living systems and the environment.
Nanotechnology deals with substances of sizes in the range of 0.1 to
100 nm. The size of most atoms lies in the range of 0.1 to 0.2 nm, the width
of DNA molecules is about 2 nm, the characteristic size of blood cells is
about 7500 nm, and the diameter of human hair is about 80000 nm. Indeed,
nanoparticles are currently the smallest chemical entities being applied in
chemical technology. Depending on the context, nanomaterials can be a single atom or an organic molecule more than 1 μm (10–6 m) in size, and con-
taining more than 109 atoms. The chemical and biological activity of nanoparticles varies significantly with difference in size. Gold and silver, in
recent times, have attracted considerable attention in biocatalysis, optics, biosensing, medical imaging and antimicrobial applications. The inertness of
gold and silver is well-known; however, clusters of several atoms of these
elements exhibit unique catalytic properties. However, for instance, catalytic
properties are observed in gold nanoparticles, composed of 8 or 22 atoms but
not in particles, consisting of 7 or 20 atoms.
S = 4 × π × r2
V = 4/3 × π × r3
Fig. 1.3. Surface area (S) and Volume (V) of a sphere
Deducing from Fig. 1.3, the surface area of a sphere is directly proportional to the square of its radius, and its volume – to the cube of its radius.
Thus, the surface area-to-volume ratio of a particle increases with decreasing
particle size. Due to the extreme small size of a nanoparticle, its internal part
is close to its surface, on which chemical reactions occur. At distances less
than 100 nm between interacting particles, their optical, electrical, magnetic
and other properties are influenced by quantum effects, making nanoparticles
behave as waves.
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