Добавил:
ivanov666
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Fundamentals of Toxicology. Tutorial
.pdf
41
– dissociation constant: dissociated molecules or ions penetrate poorly
through ion channels and do not penetrate lipid barriers;
– chemical properties: these determine the affinity of a toxicant to biochemical components of cells, tissues and organs.
Fig. 3.2. Cross-sectional scheme of toxicant transport in the human body
Toxicokinetics involves the bridging of biological barriers by chemical
substances and their distribution between biological compartments.
The properties of biological compartments of the human body include:
– the proportion of water and lipids in cells, tissues and organs: biological structures may contain either little amount of lipids (e.g. muscle tissue)
or a high amount of lipids (e.g. biological membranes, adipose tissue, brain);
and
– the presence of biomolecules that actively bind toxicants; for example, protein motifs in the skeletal system that actively bind calcium and other
divalent metals, such as lead and strontium.
3 . 2 . T r a n s p o r t o f t o x i c a n t s a c r o s s b i o m e m b r a n e s
The entry, absorption, distribution, metabolism and excretion of toxicants into and from biological systems involve the penetration (transport)
42
of the toxicants across a number of biological membranes (biomembranes).
Biomembranes have a similar phospholipid bilayer structure (Fig. 3.3) but
differ in functional properties, as well as permeability to various substances.
They are characterized by their thickness, the presence and size of pores in
them, and the presence or absence of mechanisms for active, passive or facilitated transport of chemical substances across them. The external phospholipid layer has different oligosaccharides attached to integral and peripheral membrane proteins, forming glycoprotein receptors. Similarly, some oligosaccharides bind with lipid molecules within the biomembrane external
lipid layer, forming glycolipid receptors. These receptors function as specific
sites of interaction with xenobiotics, and also play an immunofunctional role
in the recognition of foreign cells, such as bacteria, parasites and viruses.
The phospholipid molecules are oriented in such a way that their hydrophobic tails are in contact, while their hydrophilic groups are directed towards
the external and internal environment of the cell. Ultramicroscopic pores
(channels) formed by proteins, having definite electrical charges, are responsible for the active transport of substances into and out of the cell.
Fig. 3.3. Molecular structure of biomembrane: 1 – glycoprotein;
2 – glycolipid; 3 – peripheral membrane protein; 4 – integral membrane
protein; 5 – cholesterol; 6 – channel protein; 7 – phospholipid bilayer
Mechanisms of transport of substances across membranes are influenced not only by the functional features of the membranes themselves, but
also by functions of the protoplasm and cellular proteins. These mechanisms
are categorized into four main types:
Fig. 3.4. Simple diffusion across a biomembrane, separating two
compartments of different substance concentrations: the substance diffuses
over time until equilibrium is reached
Type I is characteristic of neutral molecules. In this case, molecules of
substances with a high oil/water partition coefficient, i.e., having high lipophilic properties, diffuse the fastest across membranes. Lipid-soluble substances (for example, many narcotic substances) can freely pass through
biomembranes with minimal expenditure of energy according to Fick’s laws
of diffusion. The rate of diffusion (Rd) of a substance, according to Fick’s
first law, is determined by the equation:
,
А
К
where К is coefficient of diffusion of the substance; А is area of membrane;
(C1 – С2) is concentration gradient on both sides of the membrane; and d is
thickness of membrane. The coefficient of diffusion of a toxicant depends
on its molecular weight, degree of lipid solubility and ionization, as well
as molecular spatial configuration. Small hydrophobic molecules readily
move across phospholipid bilayers by simple diffusion (Fig. 3.4; Fig. 3.6).
Large molecules, that are unable to diffuse freely across biomembranes are
transported through relatively large pores, called channel proteins
(Fig. 3.3) or by means of endocytosis (Fig. 3.5), where a vacuole containing the molecules is embedded into the membrane and its contents pushed
into the cell.
43
44
Fig. 3.5. Forms of endocytosis
Type II transmembrane transport is associated with interactions be-
tween membrane lipids, proteins and carbohydrates to provide substances
with more intense diffusion known as facilitated diffusion (Fig. 3.6).
The transported molecule reversibly binds with a carrier molecule, which
moves freely (oscillates) between the membrane’s inner and outer surfaces.
Glucose transporters, as an example, are membrane proteins that facilitate
the transport of glucose across the plasma membrane.
Fig. 3.6. Types of membrane transport
Type III transmembrane transport is accompanied by consumption of
energy; i.e., adenosine triphosphate (ATP). A molecule of the substance to
be transported binds with a carrier protein, causing the protein to undergo
chemical transformations. Examples of type III mechanism include processes of potassium (K+) and sodium (Na+) ion channel transmembrane
transport in mammalian cells, as well as absorption and excretion of
45
substances in their ionized forms by renal tubules. The biochemical mechanisms for the active transport of these ions involves the catalysis of
dephosphorylation of ATP (Fig. 3.6) by potassium and sodium-dependent
enzyme, adenosine triphosphatase (ATPase).
Type IV transport relates to diffusion through pores with positively
charged particles that allow only anions to pass through. There are also channels that allow non-electrolytes to pass through. The maximum size of these
channels correspond to the size of the largest molecule that can pass through
them. For example, human renal glomeruli membrane pores are very small,
and only albumin molecules, the smallest of proteins with molecular weight
of 66.5 kDa, have a chance of being filtered through. In membranes of this
type, the transport of substances is carried out by the principle of filtration;
only substances of a particular size can pass through the membrane depending
on the size of the membrane pores.
Chemical substances, characterized by specific membrane-toxic effects
are referred to as membranotoxins. Some natural poisons, such as tetro-
dotoxin, found in fish of the order tetraodontiformes and batrachotoxin in cer-
tain species of beetles, birds and frog, impair the function of sodium channels,
resulting in paralysis and death. Exogenous and endogenous substances with
phospholipase activity can also disorganize and break the main fluid crystal
structure of membranes, followed by cell death. Acute poisoning can cause
membrane damage through lipid peroxidation, by which permeability of
biomembranes is increased for H+ (or OH-), K+, Na+, and Ca2+ ions. This consequently disrupts osmotic function of membranes, resulting in membrane
rupture, and thus, cytolysis. The alteration to permeability to ions results in
changes in the surface charge, as well as degree of hydrophobicity of the lipid
phase of the membrane. The final change in membrane permeability varies
from case to case, having nonspecific effect on the transport of various compounds. Many chemical substances, ultraviolet rays and radiations, hyperoxia and hypoxia, hormonal disorders and stresses, vitamin deficiencies,
high and low temperature effects, immunological disruptions and pathogenic
factors can also adversely affect the membrane structure of cells.
3 . 3 . T o x i c o k i n e t i c p r o c e s s e s
The route of entry of a substance into the body is largely determined by
its state of aggregation, localization in environmental components, as well as
46
the surface area exposed to the substance (Table 3.1). For instance, a gaseous
substance has a very high probability of entry into the body through the respiratory tract; however, if dissolved in water, its main route of entry can be
the gastrointestinal tract, and less likely the skin. Factors influencing the rate
and mechanisms of entry of a substance into the body include:
– individual characteristics: age, gender, body weight, etc.;
– properties of the substance: molecular weight and size, chemical
structure, physicochemical properties, state of aggregation, etc.;
– environmental parameters: temperature, humidity, etc.;
– quantitative characteristics: contact or exposure time, concentra-
tion, dose.
Table 3.1
Surface area of the human body
Body surface
Area, m2
Skin
Oral cavity
Stomach
Small intestine
Large intestine
Rectum
Nasal cavity
Lungs
1.2–2.0
0.02
0.1–0.2
100
0.5–1.0
0.04–0.07
0.01
70
After absorption into the blood, substances are distributed unevenly
along their concentration gradient to all organs and tissues with varying retention times. Strontium and lead are metals that are in some respects close
to calcium, and thus, can gradually replace calcium in tissues. Due to chemical affinity, and depending on age, these metals mainly get deposited and
accumulated in skeletal tissue with chronic exposure. Strontium inhibits
the differentiation of osteoclast cells and increases the process of apoptosis
in mature osteoclasts, ultimately resulting in the reduction of bone resorption
properties. Apart from the toxic consequences that the accumulation of lead
may have for bone status, the effect on bone mineral metabolism extends to
organs, such as the brain, liver and kidneys.
Water-soluble high-molecular weight compounds, such as peptide toxicants, circulating in the blood, will not diffuse into tissues if the radius of
their molecules exceeds the pore radius of capillary walls. The same intravascular distribution is characteristic of even low molecular weight
47
substances that bind to plasma proteins or form large agglomerates of particles in blood. The nature of blood supply to organs determines the distribution of toxicants in the first minutes or hours after absorption until equilibrium is attained. Absorbed substances immediately get into organs through
enriched blood supply to them. These substances are further redistributed,
based on tissue properties, such as lipid and water composition, the presence
of specific receptors for some xenobiotics, and the retention capacity for specific substances. For example, in the first minutes of intravenous administration of 25 mg/kg of thiopental to a dog, the substance is detected in large
quantities in the liver (up to 90 %) but practically absent in adipose tissue.
However, the liver and adipose tissue are found to contain approximately
the same amount of the substance (up to 30 %) after three hours, a phenomenon attributed to redistribution of the substance in the organism.
Fat-soluble substances, such as barbiturates and polyhalogenated aromatic hydrocarbons (PAHs), accumulate mostly in tissues that are rich in lipids; e.g. the central nervous system (CNS) and adipose tissue. Generally,
a normal human adult body has adipose tissue of 15 to 20 % of body weight.
Fat-soluble substances have high partition coefficient values in oil/water systems, and poorly move from lipid to aqueous phase. In this regard, a number
of fat-soluble toxicants; including dioxins, halogenated dibenzofurans, and
clofenotane (DDT), have high retention times in the body, particularly, in
the nervous system and adipose tissue, but are found in low concentrations
in blood plasma. In situations of drastic decrease in body fat, free molecules
of toxicants that had been accumulated in the fat-rich tissues are released into
the bloodstream. A toxicant that enters the bloodstream can interact with proteins and blood cells. Human blood plasma contains about 75 mg/ml of proteins; the bulk of which is albumin, performing transport, nutrient absorption
and blood pressure regulatory functions. Other proteins include blood coagulation factors, immunoglobulins, enzymes, lipoprotein and glycoprotein
complexes, etc. The red blood cell also contains the oxygen-transport protein – hemoglobin, as well as the intermediary oxygen-transfer protein – myoglobin. The functions of these proteins may be impaired due to their interactions with toxicants, leading to toxic effects, such as blood coagulation,
immune system breakdown, hemolysis, and total destruction of the proteins
by denaturation.
48
3 . 4 . Q u a n t i t a t i v e c h a r a c t e r i s t i c s o f t o x i c o k i n e t i c s
The amount of a substance in the blood, as well as other biological fluids, is an important measure of its toxic effect. Thus, the values of toxic concentrations in the blood for substances of various pharmacological, as well
as toxicological categories can differ in several magnitudes (Table 3.2).
The study of the toxic effect of a chemical substance in the body involves
the use of data on the concentration of the substance or its metabolites in biological samples at specific time intervals after a dose of the substance is administered. In this regard, complexity of the human body as a biochemical system does not exclude application of average kinetic parameters for the assessment of mechanisms of metabolic reactions, using mathematical models.
Table 3.2
Therapeutic, toxic and lethal blood concentrations of some drugs
Drug substance
Concentration, mg/L
Therapeutic
Toxic
Lethal
Paracetamol
10–20
400
1500
Aspirin
20–100
150–300
500
Phenobarbital
10
40–60
80–150
Chlorpromazine
0.5
1–2
3–12
Diphenhydramine
0.06–0.14
1–8
9.2
Nitrazepam
0.04–0.12
0.2
2.9–5.0
The concentration curves in Fig. 3.7 typically characterize the simultaneous course of the processes of absorption, distribution and excretion of
chemical substances in the blood. The analyses of toxicokinetic parameters
of xenobiotics enable the evaluation, comparison, and prediction of metabolic processes or pathways of these xenobiotics, and thus establishing safety
protocols and standards for their use.
The administration of a chemical substance at specific, equal intervals of
time results in fluctuations around the average value of concentration (Cav) of
the substance in systemic blood (Fig. 3.8). The optimal range of drug concentration in the blood is the interval between the effective minimum concentration (С
ss
min
) and the maximum safe concentration (С
ss
max
). This is referred to
as safety corridor of a drug, and represented as С
ss
min
< Cav < С
ss
max
.
The concentration of substances and their metabolites in biological fluids, such as blood, urine, and saliva can be determined by chromatographic,
enzyme-linked immunoassay, and spectrofluorimetric methods.
49
Fig. 3.7. Kinetic curves of substances in blood (a) and their
semi-logarithmic plots (b); intravenous (1) and oral (2) administration
Fig. 3.8. Relationship of substance concentration in blood to time
with repeated doses (D) at regular time intervals (τ)
In processing kinetic curves of toxicants, the relationship of blood concentration to time is usually subject to 1st order kinetics. In this regard,
where
is the rate of toxicant excretion; is the excretion rate constant;
and “C” is the blood concentration of the toxicant. The relationship implies
that the rate of excretion at any given time is proportional to the blood concentration of the substance. The measure of the rate of excretion of a substance is determined by the value of the angle of inclination of the tangent to
the curve at a particular point of interest or by the value of
. The rate of
excretion decreases over time as the value of “C” decreases.
50
Integrating the equation, we have
where C0 is initial concentration of the substance; Ct is concentration of
the substance at a particular time; “t” is time after administration of the substance; and KE is the excretion rate constant.
After the determination of the value of KE, calculation of an important
toxicokinetic parameter – the half-life (t
1/2
) of an administered drug substance, i.e. the time during which half of the substance is excreted from
the body, can easily be done as follows:
Bioavailability – another toxicokinetic parameter – can be calculated,
based on the value of the area under the kinetic curve. Bioavailability is
the ability of a substance in a particular aggregate state, bound to inert carriers (soil, food, solvent), to be absorbed into the systemic circulation of
the body, and thus, reach targets of interaction with corresponding receptors.
Taking into consideration the route of entry of a substance, the value of its
bioavailability increases with increasing area under curve (AUC). Similarly,
the toxic effect of a xenobiotic is directly proportional to its AUC. The larger
the area under the kinetic curve for an administered dose of a xenobiotic,
the longer it takes for the substance to be excreted from the body.
3.5 . B i o t r a n s f o r m a t i o n a n d e x c r e t i o n
of x e n o b i o t i c s
The preservation of chemical homeostasis is ensured by different mechanisms, involving various organs and organ systems of the body. Most xenobiotics undergo partial or complete chemical transformation in two phases
(Fig. 3.9) before excreted through various pathways from the body. The process of excretion, in order of practical significance, is carried out by the kidneys, gastrointestinal tract, lungs, and skin. However, some compounds are
excreted without transformation. At physiological pH, the ionization of xenobiotics increases with increasing solubility in water, but decreasing constructive transformation. The extent of biotransformation of a xenobiotic
Соседние файлы в предмете [НЕСОРТИРОВАННОЕ]