Kinetics of Chemical Compounds

The kinetic properties of chemical compounds include their absorption and distri­bution in the body, their biotransformation to more soluble forms through meta­bolic processes in the liver and other metabolic organs, and the excretion of the metabolites in the urine, the bile, the exhaled air, and in the saliva. An important issue in toxicokinetics deals with the formation of reactive toxic intermediates during phase I metabolic reactions (see Section 5.3.3).

5.3.3.1 Absorption

As stated earlier, inhalation is the main route of absorption for occupa­tional exposure to chemicals. Absorption of gaseous substances depends on solubility in blood and tissues (as presented in Sections 2.3.3-2.3.5), blood flow, and pulmonary ventilation. Particle size has an important influence on the absorption of aerosols (see Sections 2.3.7 and 3.1.1).

Absorption via the skin depends on the lipid — and water-solubility of the compound, its polarity, and the molecular size. Dermal absorption is also markedly affected by the size of the exposed skin area.52’54’65’67

Chemicals have to pass through either the skin or mucous membranes lining the respiratory airways and gastrointestinal tract to enter the circulation and reach their site of action. This process is called absorption. Different mechanisms of entry into the body also greatly affect the absorption of a compound. Passive diffusion is the most important transfer mechanism. According to Fick’s law, 57 diffusion velocity v depends on the diffusion constant (D), the surface area of the membrane {A), concentration difference across the membrane (Ac), and thickness of the membrane (L)

The diffusion constant depends on the lipid solubility, molecular weight, and structure of the substance. Lipid-soluble compounds with a molecular weight less than about 500 diffuse readily through the membranes. Polar compounds are poorly absorbed. However, active transport systems play a major role in the ab­sorption of a number of amino acids, sugars, ions, and other nutrients. The blood — brain barrier, a functional structure that protects the central nervous system (CNS) against foreign substances, prevents the entry of most compounds in to the CNS. In fact, only lipid-soluble compounds, and polar compounds which have an active transport mechanism, can readily enter the CNS. Examples of such polar com­pounds include amino acids and sugars. Active transport also plays an important role in the testicles, which are protected by a testicular-blood barrier which has a role similar to the blood-brain barrier. j7>68 Figure 5.34a shows how mevinphos, a greenhouse organophosphorus insecticide which mainly gains access to the body via the skin, is absorbed. Figure 5.34b shows that exposure through lungs was neg­ligible, because there was an excellent correlation between mevinphos on the foli­age, the source of the compound, and mevinphos level on the skin. 34

Mevinphos (ng/cm2)

Time (h) after application (a)

Mevinphos (ng/m3)

FIGURE 5.34 (a) Correlations (y = 7.2x S 3.5; r = 0.97) between the amount of mevinphos on The foliage and the dermal exposure rate to mevinphos via the hands.5“1 (b) Mean (±SD) concentrations of mevinphos in the breathing zone of the workers immediately after application and on the morning of the two first working days after the application.^ (Used with permission.)

(b)

Entry of Particles into the Body

The aerodynamic particle diameter determines the fate of particles in the respiratory system. Coarse particles are deposited in the nose and nasophar­ynx. Smaller particles that pass the upper airway can be deposited in the bron­chial region and lower airway. A size-selective deposition model and sampling of particles has been standardized both in Europe69 and internationally.70 The
standard includes size definitions for three mass fractions. The inhalable frac­tion consists of particles that can enter the upper airway. Its upper size limit is 100 p, m (the diameter of human hair is 50-100|xm). The thoracic mass frac­tion consists of particles that can penetrate past the larynx. Its upper size limit is about 30 ijum and median cut point 10 |xm. The respirable mass fraction consists of particles that can enter the alveolar region. Its upper size limit is about 10 |juti and median cut point 4 |j. m (see Fig. 5.3.5).

5.3.3.2 Distribution

After absorption, a chemical compound enters the circulation, which transfers it to all parts of the body. After this phase, the most important factor affecting the distribution is the passage of the compound through biological membranes. From the point of view of the distribution of a chemical com­pound, the organism can be divided into three different compartments: (1) the plasma compartment; (2) the intercellular compartment; and (3) the intracel­lular compartment. In all these compartments, a chemical compound can be bound to biological macromolecules. The proportion of bound and unbound (free) chemical compound depends on the characteristics of both the chemical and the binding macromolecules.63,64

In the plasma, most chemicals are bound to plasma proteins. Albumin is quantitatively the most important binding protein but beta globulin and acidic glycoprotein also bind chemicals. The number of binding sites is lim­ited, and, therefore, high doses of chemicals may cause saturation of protein binding. In most cases, an adverse effect does not require saturation of pro­tein binding sites because free and bound chemical are in equilibrium in the plasma, and the free chemical is available for toxic action in the target tis­sues. The circulation is extremely important for distribution of chemicals. Heavily perfused organs, i. e., the brain, liver, and kidneys, receive most of the cardiac output, and in these organs the concentration of a chemical in­creases much more rapidly than in the other organs. Organs whose perfu­sion is small, e. g., resting muscles and adipose tissue, receive only a small portion of the cardiac output, and therefore concentration of a chemical in these organs increases much more slowly than in the heavily perfused or­gans.62-64

Nasopharyngeal region 5 -30 jim

Bronchius, bronchial, and bronchiolar region 1-5 (im

Alveolar region 1 ii m

FIGURE 5.35 Regions of pulmonary pathways and size of particles that can reach different regions of the lungs.4

Adipose tissue and bones function as storage sites for many substances. Most chemicals have some tissue specificity with regard to their tissue binding. In many cases, this property of a chemical is not important, but, especially for lipid-soluble chemicals, adipose tissue often becomes an important storage de­pot from which they are slowly released. Both accumulation and reler. se of compounds from the adipose tissue are slow processes, partly because adipose tissue receives only 2% of the cardiac output. The accumulated compound may­be released if the size of the fat depot decreases. For example, lipid-soluble in­secticides, such as chlordane, may even cause acute intoxication due to dieting, and dieting also causes release of the supertoxic compound dioxin into the cir­culation. Lipid-soluble compounds can also be released from their depots in the adipose tissue during breast feeding of infants, and this may cause excessive ex­posure.65,71 The features of absorption, distribution, and excretion have been depicted in Fig. 5.36.

Another important storage depot for toxic compounds is the skeleton. In particular, cadmium and lead bind and accumulate in the bone tissue from which they are released very slowly. The half-life of elimination of cadmium is several years, the half-life of lead is several months.

Theoretical volume of distribution (Vd) of a chemical is the volume in which the chemical would be distributed if its concentration were equal to a theoretical steady-state plasma concentration (C0) at time zero. The volume of distribution is thus obtained quite similarly as the steady state concentration of a compound in the workroom air:

Vd=F-, (5.53)

‘-‘0

Where m is the mass of a chemical and C0 is its theoretical plasma concentration at time zero. Even though the compound does not ever reach the theoretical

Volume of distribution in practice, it provides valuable information on the char­acteristics of the compound, that can be used in a number of pharmaco/toxico­kinetic models.62’68

Special Considerations

Chemical compounds may also be distributed to the placenta and through the placenta to the fetus and thereby cause exposure of the offspring. Even though the placental wall consists of several layers, it is a biological mem­brane, and the same principles apply to the placenta as to any other biological membrane, i. e., penetration depends on lipid-solubility and ionization of chemical compounds. However, the concentrations of lipid-soluble com­pounds increase slowly in the fetus, because it is a separate body compart­ment, and redistribution with this compartment is a time-consuming process. However, an equilibrium will be reached between the mother and the fetus during long-term exposure. This is the main reason chemical exposure during pregnancy is strictly controlled in most developed countries.

5.3.3.3 Biotransformation

The purpose of metabolism or biotransformation of xenobiotics (foreign compounds) is to transform them into a water-soluble form so that they can be excreted either in the urine or in the bile. These processes are catalyzed by a number of enzymes. Biotransformation reactions are divided into phase I and phase II reactions: in phase I reactions, functional groups, such as the hy­droxyl group, are linked to the xenobiotic (Fig. 5.37). This is why phase I re­actions are also called functionalization reactions. In phase II, the functional group is conjugated with one of several chemical compounds in the body, e. g., glucuronic acid, glutathione, sulfates, glycine, or methionine. Most xenobiot­ics undergo both phase I and phase II reactions, but some compounds undergo only one of the phases. It is noteworthy that rarely will all of the absorbed

FIGURE 5.37 Janus faces of the biotransformation of xenobiotics. On one hand metabolism leads to inactivation and elimination of xenobiotics, but on the other hand many metabolites are reactive and may cause deleterious effects by binding to DNA, proteins, and other macromolecules.

Compound be metabolized; in most cases small amounts of unchanged parent compound can also be found in the urine. This can also be utilized us a spe­cific biological monitoring test. The enzymes responsible tor biotransforma­tion of xenobiotics also catalyze the metabolism of endogenous compounds, such as hormones and neurotransmitters. For example, steroid hormones un­dergo phase I oxidation catalyzed by P450 enzymes and then conjugation re­actions of the functional groups catalyzed by glucuronyltranslerase or sulfotransferase. The number of possible metabolites of various chcinicais is often very large because of the variety of different phase I and phase tl en­zymes in the cells.63,72

The highest activities and amounts (the amount of enzyme protein/g pro­tein in the tissue) of biotransformation enzymes are found in the liver, and this organ plays a key role in the metabolism of endogenous and foreign com­pounds. However, these enzymes can be found in many other organs, and one can hypothesize that enzymes expressed at the entries to the body, i. e., the skin and the mucosa of the gastrointestinal tract and airways, have developed dur­ing evolution to protect the organism against foreign compounds, In fact, the liver and kidneys are also direct or indirect sites of entry of foreign com­pounds into the body. The liver is an important port of entry because of the portal vein that carries most foreign compounds directly from the intestine to the liver. The kidneys can also be a port for chemical compounds into the body because a number of compounds excreted in the urine may be reab­sorbed in the proximal tubules of the kidney. Such compounds include those with an active transport system, many lipid-soluble compounds, and metabo­lites that have been hydrolyzed in the urine. On the other hand, metabolites of these parent compounds conjugated with biological macromolecules or amino acids are readily excreted in the urine or bile.68,72

Biologically active compounds are often inactivated during phase I biotransformation. However, in some instances, biological activity of chemical compounds may be increased, and they can become activated especially by the P450 enzyme (CYP enzymes) system that catalyzes oxidation, hydroxylation, and epoxidation. These reactions may yield electrophilic intermediates that readily react with the nucleophilic groups of biological macromolecules, such as nucleic acids and proteins, with toxic consequences, such as cell death, mu­tagenesis, malignant transformation of cells, or teratogenesis. For example, activation of carbon tetrachloride, bromobenzene, and acetaminophen (para­cetamol) after high doses cause liver necrosis. At lower doses, they may cause genotoxic alterations in the cells and subsequent malignant transformation of the exposed cells. Active metabolites of aflatoxin B1 (a fungal toxin), benzo(a)pyrene (a combustion product), and vinyl chloride (a plastic mono­mer), induce cancer subsequent to their binding with bases in the DNA. Since all of the compounds that are absorbed in the gastrointestinal tract enter the liver directly through the portal vein, their biotransformation is very effective in the liver because of their extensive contact with metabolically active liver cells, the hepatocytes. The anatomical structure of the liver further promotes effective biotransformation of xenobiotics in this organ (Fig. 5.38). A number of factors affect the metabolizing capacity of the liver. These include the con­centration of phase I CYP enzymes, the uptake of the compounds bv the lier

FIGURE 5.38 Pictorial presentation of the microscopic structure of the liver. The picture shows the classical liver lobulus. The functional acinus and its three zones are at the left. The acinal zones are marked by numbering them 1-3. These zones correspond to the direction of blood flow from the portal arteries (PA) to the terminal veins (TV). Zone I corresponds to the periportal area in classical liver pathology, zone 2, the interlobular region (midzone), and zone 3, centrelobular region.71

Cells, blood flow through the liver, and different pathological processes such as collagen formation due to cirrhosis and hepatitis. ’5’56

5.3.3.4 Excretion

Water solubility (polarity) is essential for excretion. Even though lipid-sol­uble compounds may also be excreted to primary urine, they are usually at least partially reabsorbed. The metabolites formed in the liver and extrahe — patic tissues remain free (i. e., not bound to proteins) and are, therefore, readily excreted.

Cadmium is effectively accumulated in the kidneys. When the cadmium concentration exceeds 200 |xg/g in the kidney cortex, tubular damage will oc­cur in 10% of the population, and proteins begin to leak into urine (pro­teinuria). When the concentration of cadmium in the kidney cortex exceeds 300 p. g/g, the effect is seen in 50% of the exposed population. Typically, ex­cretion of low-molecular weight proteins, such as beta-microglobulin, is in­creased, due to dysfunction of proximal tubular cells of the kidney. The existence of albumin or other high-molecular weight proteins in the urine indi­cates that a glomerular injury has also taken place. The excretion of protein — bound cadmium will also be increased.62’63-73

Pulmonary excretion takes place for volatile compounds. Alveolar air is at equilibrium with capillary blood. Thus, pulmonary excretion depends on the vapor pressure of the compound and its blood solubility. If blood solubility is

Low, the compound will be rapidly excreted (see Section 2.3.9). The determi­nation of alveolar air concentration can be used as biological exposure test for organic solvents. This test is also widely applied to control for drunken driv­ing. The concentration of a solvent in the blood is obtained by multiplying the alveolar air concentration by the blood solubility coefficient.6- 74

Lungs also secrete nonvolatile compounds. Lipid-soluble compounds may thus be transported with the alveobronchotracheal mucus to the pharynx, where they are swallowed. They may then be excreted or reabsorbed. Particles are also removed by this mucociliary escalator.

The particle size is the most important factor that contributes to the clear­ance of particles. For particles deposited in the anterior parts of the nose, wip­ing and blowing are important mechanisms whereas particles on the other areas of the nose are removed with mucus. The cilia move the mucus toward the glottis where the mucus and the particles are swallowed. In the tracheo­bronchial area, the mucus covering the tracheobronchial tree is moved up­ward by the cilia beating under the mucus. This mucociliary escalator transports deposited particles and particle-filled macrophages to the pharynx, where they are also swallowed. Mucociliary clearance is rapid in healthy adults and is complete within one to two days for particles in the lower air­ways. Infection and inflammation due to irritation or allergic reaction can markedly impair this form of clearance.

Particles deposited in the alveoli are phagocytized by alveolar macroph­ages and cleared either through the mucociliary escalator or through the lym­phatic drainage system. Fibers may be too long to become phagocytized by single macrophages. In such a case, several macrophages can participate in the phagocytosis in a cooperative manner (see Fig. 5.39). Macrophages are able to dissolve synthetic miner fibers to some extent but asbestos (especially amfi- boles) fibers remain mostly unaffected, This leads to the production of oxygen radicals and inflammation mediators which induce macrophages to kill them­selves. Another macrophage will then phagocytize the asbestos fiber and it too will die. This vicious cycle will continue and it may ultimately lead to lung fi­brosis and cancer. Small particles may also directly penetrate the epithelial membrane and enter the blood stream.

5.3.3.5 Movements of Chemical Compounds in the Body

Absorption, distribution, biotransformation, and excretion of chemical compounds have been discussed as separate phenomena. In reality all these processes occur simultaneously, and are integrated processes, i. e., they all af­fect each other. In order to understand the movements of chemicals in the body, and for the delineation of the duration of action of a chemical in the or­ganism, it is important to be able to quantify these toxicokinetic phases. For this purpose various models are used, of which the most widely utilized are the one-compartment, two-compartment, and various physiologically based pharmacokinetic models. These models resemble models used in ventilation engineering to characterize air exchange.

One-Compartment Model

The simplest toxicokinetic analysis involves measurement of the plasma concentrations of a chemical at several time points after the administration of

Asbestos fibers FIGURE 5.39 Possible pathways in fiber carcinogenesis. The figure is based on review articles by 1992 and Kamp et al. 1992, and original publications by Marsh and Mossman 1991,76 Heintz et al. 1993, Kodama et al. 1993, Kinnula et al. 1994b, as well as the results presented in studies II and V. The connection between disrupted cytoplasmic division, cytoskeletal changes, and decreased intercellular adhesion has not been studied in relation to fibers. The pathway is based on articles dealing with link­age between cadherins, catenins, and cytoskeleton, as well as their role in cell transformation.73"80 The connection between decreased cell-cell adhesion and decreased GJIC has been reported by Musil et al.81 and Meyer et al.,82 and the role of GJIC in cell transformation has been reviewed by Yamasaki,8! (modified from Pelin).84

A single intravenous injection. If the kinetic data obtained yield a straight line when plotted as the logarithm of plasma concentrations versus time, the ki­netics of the compound can be described by a one-compartment model, in which the whole body is treated as one single space or compartment. Even though the one-compartment model is an extreme simplification of the or­ganism in the physiological and toxicological sense, the behavior of several chemical compounds can be well described and understood by using this model. Usually, only the kinetics of compounds that are rapidly distributed in the body can be described with the one-compartment model. As described be­low (Fig. 5.40), the theoretical concentration C0 can then be calculated.

The rate of elimination of a chemical compound from the body is usu­ally proportional to the amount of the chemical in the body. Elimination processes include biotransformation, exhalation, and excretion in the urine, bile, saliva, and sweat, and even in the hair and nails. The first-order

Elimination rate constant kei has units of reciprocal time (e. g., mui and h_1). For example, if the elimination rate constant is 0.5 h-1 the percentage of the dose excreted after one, two, or three hours is the same, regardless of the given dose. In this case, the percentage of the dose excreted is 43 !o, even though the rate constant is 0.5/h (or 50%/h), because the dose re­maining in the body (C) decreases continuously with time. The elimination rate decreases ( kei ■ C ) when the dose remaining in the body (Cl decreases. The first-order elimination rate of the compound is mathematically ex­pressed as an exponential equation C = Cq ■ exp(-fep;/:) where C is the plasma concentration, kei the first-order elimination rate constant, and t the time of blood sampling. With logarithmic transformation a straight line is obtained:

In C, = In C0 — kei ■ t (5.54)

Where In C0 represents the intercept and — ke( represents the slope of the line. Therefore, the first-order elimination rate constants can be determined by uti­lizing the slope of the In C versus time plot.

In addition to the elimination rate constant, the half-life (Zj/2) is an­other important parameter that characterizes the time-course of chemical compounds in the body. The elimination half-life (J1/2) is the time to re­duce the concentration of a chemical in plasma to half of its original level. The relationship of half-life to the elimination rate constant is ti/2 = 0.693/kei and, therefore, the half-life of a chemical compound can be determined after the determination of kej from the slope of the line. The half-life can also be determined through visual inspection from the log C versus time plot (Fig. 5.40). For compounds that are eliminated through first-order kinetics, the time required for the plasma concentra­tion to be decreased by one half is constant. It is important to understand that the half-life of chemicals that are eliminated by first-order kinetics is independent of dose.68’85’86

Two-Comportment Model

If the plotting of the logarithm of the plasma concentration against time does not result in a straight line but rather in a curve, the use of multicompart­ment models is required. Multicompartment models are required for com­pounds that distribute to different organs at different rates. Such compounds are usually lipid-soluble and reach equilibrium in lipid-containing organs rela­tively slowly. This results in multiexponential elimination because chemical compounds are eliminated in a reverse order as compared with their distibu — tion. In the simplest case, this type of curve can be resolved into two exponen­tial terms (a two-compartment model). Concentration can be expressed as C = Aea + BP where A and B are proportionality constants and a and [i are rate constants with dimensions of reciprocal time. During the distribution al­pha phase, concentrations of the chemical in plasma decrease more rapidly than they do in the postdistribution phase (beta). The length of the distribu­tion phase may vary from minutes to hours to days. Whether the distribution phase becomes apparent depends on the time of the sampling after the cessa­tion of the exposure. Since most chemicals in the occupational environment

FIGURE 5.40 Schematic representation of the concentration of a chemical in the plasma as a func­tion of time after an intravenous injection if the body acts as a one-compartment system and elimination of the chemical obeys first-order kinetics with a rate constant (ke,).48

1003

Follow two — or other multicompartmental elimination kinetics, the correct timing of blood sampling for biological monitoring is essential.68’85’86

In a two-compartment model, ji is equivalent to k in the one-compartment model. Therefore, the terminal half-life for the elimination of a chemical com­pound following two-compartment model elimination can be calculated from the equation /3 = 0.693/ti/2 .

Saturation of Elimination

Saturation kinetics are also called zero-order kinetics or Michaelis­Menten kinetics. The Michaelis-Menten equation is mainly used to character­ize the interactions of enzymes and substrates, but it is also widely applied to characterize the elimination of chemical compounds from the body. The sub­strate concentration that produces half-maximal velocity of an enzymatic re­action, termed Km value or Michaelis constant, can be determined experimentally by graphing vi as a function of substrate concentration, [5].

System that may become saturated follows zero-order kinetics. As the concentra­tion of a chemical compound decreases to below the concentration at which the enzyme becomes saturated, it changes to first-order kinetics.

From a practical point of view, saturation of elimination has important consequences. If the metabolism becomes saturated, the duration of the ac­tion of the compound is prolonged. In such a case, correct timing for collec­tion of biological monitoring samples also becomes difficult to assess. Furthermore, saturation of metabolism may also have qualitative effects. For example, it has been argued (but not yet proved) that arsenic compounds cause cancer at high doses at which methylation of inorganic arsenic be­comes saturated.68

Physiologically Based Toxicokinetic Models

Physiologically based toxicokinetic models are nowadays used increasingly for toxicological risk assessment. These models are based on human physiol­ogy, and thus take into consideration the actual toxicokinetic processes more accurately than the one — or two-compartment models. In these models, all of the relevant information regarding absorption, distribution, biotransformarion, and elimination of a compound is utilized. The principles of physiologically based pharmaco/ toxicokinetic models are depicted in Fig. 5.41a and b. The

(b)

Main difficulty in using these models is that in most cases not enough informa­tion is currently available about the compound under study.68 83

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