Exposure, Dose and Response

Toxicity is the intrinsic capacity of a chemical agent to affect an organism adversely.

Xenobiotics is a term for “foreign substances”, that is, foreign to the organism. Its opposite is endogenous compounds. Xenobiotics include drugs, industrial chemicals, naturally occurring poisons and environmental pollutants.

Hazard is the potential for the toxicity to be realized in a specific setting or situation.

Risk is the probability of a specific adverse effect to occur. It is often expressed as the percentage of cases in a given population and during a specific time period. A risk estimate can be based upon actual cases or a projection of future cases, based upon extrapolations.

Toxicity rating and toxicity classification can be used for regulatory purposes. Toxicity rating is an arbitrary grading of doses or exposure levels causing toxic effects. The grading can be “supertoxic,” “highly toxic,” “moderately toxic” and so on. The most common ratings concern acute toxicity. Toxicity classification concerns the grouping of chemicals into general categories according to their most important toxic effect. Such categories can include allergenic, neurotoxic, carcinogenic and so on. This classification can be of administrative value as a warning and as information.

The dose-effect relationship is the relationship between dose and effect on the individual level. An increase in dose may in- crease the intensity of an effect, or a more severe effect may result. A dose-effect curve may be obtained at the level of the whole organism, the cell or the target molecule. Some toxic effects, such as death or cancer, are not graded but are “all or none” effects.

The dose-response relationship is the relationship between dose and the percentage of individuals showing a specific effect. With increasing dose a greater number of individuals in the exposed population will usually be affected.

It is essential to toxicology to establish dose-effect and dose- response relationships. In medical (epidemiological) studies a criterion often used for accepting a causal relationship between an agent and a disease is that effect or response is proportional to dose.

Several dose-response curves can be drawn for a chemical—one for each type of effect. The dose-response curve for most toxic effects (when studied in large populations) has a sigmoid shape. There is usually a low-dose range where there is no response detected; as dose increases, the response follows an ascending curve that will usually reach a plateau at a 100% response. The dose-response curve reflects the variations among individuals in a population. The slope of the curve varies from chemical to chemical and between different types of effects. For some chemicals with specific effects (carcinogens, initiators, mutagens) the dose-response curve might be linear from dose zero within a certain dose range. This means that no threshold exists and that even small doses represent a risk. Above that dose range, the risk may increase at greater than a linear rate.

Variation in exposure during the day and the total length of exposure during one’s lifetime may be as important for the outcome (response) as mean or average or even integrated dose level. High peak exposures may be more harmful than a more even exposure level. This is the case for some organic solvents. On the other hand, for some carcinogens, it has been experimentally shown that the fractionation of a single dose into several exposures with the same total dose may be more effective in producing tumours.

A dose is often expressed as the amount of a xenobiotic entering an organism (in units such as mg/kg body weight). The dose may be expressed in different (more or less informative) ways: exposure dose, which is the air concentration of pollutant inhaled during a certain time period (in work hygiene usually eight hours), or the retained or absorbed dose (in industrial hygiene also called the body burden), which is the amount present in the body at a certain time during or after exposure. The tissue dose is the amount of substance in a specific tissue and the target dose is the amount of substance (usually a metabolite) bound to the critical molecule. The target dose can be expressed as mg chemical bound per mg of a specific macromolecule in the tissue. To apply this concept, information on the mechanism of toxic action on the molecular level is needed. The target dose is more exactly associated with the toxic effect. The exposure dose or body burden may be more easily available, but these are less precisely related to the effect.

In the dose concept a time aspect is often included, even if it is not always expressed. The theoretical dose according to Haber’s law is D = ct, where D is dose, c is concentration of the xenobiotic in the air and t the duration of exposure to the chemical. If this concept is used at the target organ or molecular level, the amount per mg tissue or molecule over a certain time may be used. The time aspect is usually more important for understanding repeated exposures and chronic effects than for single exposures and acute effects.

Additive effects occur as a result of exposure to a combination of chemicals, where the individual toxicities are simply added to each other (1+1= 2). When chemicals act via the same mechanism, additivity of their effects is assumed although not always the case in reality. Interaction between chemicals may result in an inhibition (antagonism), with a smaller effect than that expected from addition of the effects of the individual chemicals (1+1 2). Alternatively, a combination of chemicals may produce a more pronounced effect than would be expected by addition (increased response among individuals or an increase in frequency of response in a population), this is called synergism (1+1 >2).

Latency time is the time between first exposure and the appearance of a detectable effect or response. The term is often used for carcinogenic effects, where tumours may appear a long time after the start of exposure and sometimes long after the cessation of exposure.

A dose threshold is a dose level below which no observable effect occurs. Thresholds are thought to exist for certain effects, like acute toxic effects; but not for others, like carcinogenic effects (by DNA-adduct-forming initiators). The mere absence of a response in a given population should not, however, be taken as evidence for the existence of a threshold. Absence of response could be due to simple statistical phenomena: an adverse effect occurring at low frequency may not be detectable in a small population.

LD₅₀ (effective dose) is the dose causing 50% lethality in an animal population. The LD₅₀ is often given in older literature as a measure of acute toxicity of chemicals. The higher the LD₅₀, the lower is the acute toxicity. A highly toxic chemical (with a low LD₅₀) is said to be potent. There is no necessary correlation between acute and chronic toxicity. ED₅₀ (effective dose) is the dose causing a specific effect other than lethality in 50% of the animals.

NOEL (NOAEL) means the no observed (adverse) effect level, or the highest dose that does not cause a toxic effect. To establish a NOEL requires multiple doses, a large population and additional information to make sure that absence of a response is not merely a statistical phenomenon. LOEL is the lowest observed effective dose on a dose-response curve, or the lowest dose that causes an effect.

A safety factor is a formal, arbitrary number with which one divides the NOEL or LOEL derived from animal experiments to obtain a tentative permissible dose for humans. This is often used in the area of food toxicology, but may be used also in occupational toxicology. A safety factor may also be used for extrapolation of data from small populations to larger populations. Safety factors range from 10⁰ to 10³. A safety factor of two may typically be sufficient to protect from a less serious effect (such as irritation) and a factor as large as 1,000 may be used for very serious effects (such as cancer). The term safety factor could be better replaced by the term protection factor or, even, uncertainty factor. The use of the latter term reflects scientific uncertainties, such as whether exact dose-response data can be translated from animals to humans for the particular chemical, toxic effect or exposure situation.

Extrapolations are theoretical qualitative or quantitative estimates of toxicity (risk extrapolations) derived from translation of data from one species to another or from one set of dose-response data (typically in the high dose range) to regions of dose-response where no data exist. Extrapolations usually must be made to predict toxic responses outside the observation range. Mathematical modelling is used for extrapolations based upon an understanding of the behaviour of the chemical in the organism (toxicokinetic modelling) or based upon the understanding of statistical probabilities that specific biological events will occur (biologically or mechanistically based models). Some national agencies have developed sophisticated extrapolation models as a formalized method to predict risks for regulatory purposes. (See discussion of risk assessment later in the chapter.)

Systemic effects are toxic effects in tissues distant from the route of absorption.

Target organ is the primary or most sensitive organ affected after exposure. The same chemical entering the body by different routes of exposure dose, dose rate, sex and species may affect different target organs. Interaction between chemicals, or between chemicals and other factors may affect different target organs as well.

Acute effects occur after limited exposure and shortly (hours, days) after exposure and may be reversible or irreversible.

Chronic effects occur after prolonged exposure (months, years, decades) and/or persist after exposure has ceased.

Acute exposure is an exposure of short duration, while chronic exposure is long-term (sometimes life-long) exposure.

Tolerance to a chemical may occur when repeat exposures result in a lower response than what would have been expected without pretreatment.

Uptake and Disposition

Transport processes

Diffusion. In order to enter the organism and reach a site where damage is produced, a foreign substance has to pass several barriers, including cells and their membranes. Most toxic substances pass through membranes passively by diffusion. This may occur for small water-soluble molecules by passage through aqueous channels or, for fat-soluble ones, by dissolution into and diffusion through the lipid part of the membrane. Ethanol, a small molecule that is both water and fat soluble, diffuses rapidly through cell membranes.

Diffusion of weak acids and bases. Weak acids and bases may readily pass membranes in their non-ionized, fat-soluble form while ionized forms are too polar to pass. The degree of ionization of these substances depends on pH. If a pH gradient exists across a membrane they will therefore accumulate on one side. The urinary excretion of weak acids and bases is highly dependent on urinary pH. Foetal or embryonic pH is somewhat higher than maternal pH, causing a slight accumulation of weak acids in the foetus or embryo.

Facilitated diffusion. The passage of a substance may be facilitated by carriers in the membrane. Facilitated diffusion is similar to enzyme processes in that it is protein mediated, highly selective, and saturable. Other substances may inhibit the facilitated transport of xenobiotics.

Active transport. Some substances are actively transported across cell membranes. This transport is mediated by carrier proteins in a process analogous to that of enzymes. Active transport is similar to facilitated diffusion, but it may occur against a concentration gradient. It requires energy input and a metabolic inhibitor can block the process. Most environmental pollutants are not transported actively. One exception is the active tubular secretion and reabsorption of acid metabolites in the kidneys.

Phagocytosis is a process where specialized cells such as macrophages engulf particles for subsequent digestion. This transport process is important, for example, for the removal of particles in the alveoli.

Bulk flow. Substances are also transported in the body along with the movement of air in the respiratory system during breathing, and the movements of blood, lymph or urine.

Filtration. Due to hydrostatic or osmotic pressure water flows in bulk through pores in the endothelium. Any solute that is small enough will be filtered together with the water. Filtration occurs to some extent in the capillary bed in all tissues but is particularly important in the formation of primary urine in the kidney glomeruli.

Absorption

Absorption is the uptake of a substance from the environment into the organism. The term usually includes not only the entrance into the barrier tissue but also the further transport into circulating blood.

Pulmonary absorption. The lungs are the primary route of deposition and absorption of small airborne particles, gases, vapours and aerosols. For highly water-soluble gases and vapours a significant part of the uptake occurs in the nose and the respiratory tree, but for less soluble substances it primarily takes place in the lung alveoli. The alveoli have a very large surface area (about 100m² in humans). In addition, the diffusion barrier is extremely small, with only two thin cell layers and a distance in the order of micrometers from alveolar air to systemic blood circulation. This makes the lungs very efficient not only in the exchange of oxygen and carbon dioxide but also of other gases and vapours. In general, the diffusion across the alveolar wall is so rapid that it does not limit the uptake. The absorption rate is instead dependent on flow (pulmonary ventilation, cardiac output) and solubility (blood: air partition coefficient). Another important factor is metabolic elimination. The relative importance of these factors for pulmonary absorption varies greatly for different substances. Physical activity results in increased pulmonary ventilation and cardiac output, and decreased liver blood flow (and, hence, biotransformation rate). For many inhaled substances this leads to a marked increase in pulmonary absorption.

Percutaneous absorption. The skin is a very efficient barrier. Apart from its thermoregulatory role, it is designed to protect the organism from micro-organisms, ultraviolet radiation and other deleterious agents, and also against excessive water loss. The diffusion distance in the dermis is on the order of tenths of millimetres. In addition, the keratin layer has a very high resistance to diffusion for most substances. Nevertheless, significant dermal absorption resulting in toxicity may occur for some substances—highly toxic, fat-soluble substances such as organophosphorous insecticides and organic solvents, for example. Significant absorption is likely to occur after exposure to liquid substances. Percutaneous absorption of vapour may be important for solvents with very low vapour pressure and high affinity to water and skin.

Gastrointestinal absorption occurs after accidental or intentional ingestion. Larger particles originally inhaled and deposited in the respiratory tract may be swallowed after mucociliary transport to the pharynx. Practically all soluble substances are efficiently absorbed in the gastrointestinal tract. The low pH of the gut may facilitate absorption, for instance, of metals.

Other routes. In toxicity testing and other experiments, special routes of administration are often used for convenience, although these are rare and usually not relevant in the occupational setting. These routes include intravenous (IV), subcutaneous (sc), intraperitoneal (ip) and intramuscular (im) injections. In general, substances are absorbed at a higher rate and more completely by these routes, especially after IV injection. This leads to short-lasting but high concentration peaks that may increase the toxicity of a dose.

Distribution

The distribution of a substance within the organism is a dynamic process which depends on uptake and elimination rates, as well as the blood flow to the different tissues and their affinities for the substance. Water-soluble, small, uncharged molecules, univalent cations, and most anions diffuse easily and will eventually reach a relatively even distribution in the body.

Volume of distribution is the amount of a substance in the body at a given time, divided by the concentration in blood, plasma or serum at that time. The value has no meaning as a physical volume, as many substances are not uniformly distributed in the organism. A volume of distribution of less than one l/kg body weight indicates preferential distribution in the blood (or serum or plasma), whereas a value above one indicates a preference for peripheral tissues such as adipose tissue for fat soluble substances.

Accumulation is the build-up of a substance in a tissue or organ to higher levels than in blood or plasma. It may also refer to a gradual build-up over time in the organism. Many xenobiotics are highly fat soluble and tend to accumulate in adipose tissue, while others have a special affinity for bone. For example, calcium in bone may be exchanged for cations of lead, strontium, barium and radium, and hydroxyl groups in bone may be exchanged for fluoride.

Barriers. The blood vessels in the brain, testes and placenta have special anatomical features that inhibit passage of large molecules like proteins. These features, often referred to as blood-brain, blood-testes, and blood-placenta barriers, may give the false impression that they prevent passage of any substance. These barriers are of little or no importance for xenobiotics that can diffuse through cell membranes.

Blood binding. Substances may be bound to red blood cells or plasma components, or occur unbound in blood. Carbon monoxide, arsenic, organic mercury and hexavalent chromium have a high affinity for red blood cells, while inorganic mercury and trivalent chromium show a preference for plasma proteins. A number of other substances also bind to plasma proteins. Only the unbound fraction is available for filtration or diffusion into eliminating organs. Blood binding may therefore increase the residence time in the organism but decrease uptake by target organs.

Elimination

Elimination is the disappearance of a substance in the body. Elimination may involve excretion from the body or transformation to other substances not captured by a specific method of measurement. The rate of disappearance may be expressed by the elimination rate constant, biological half-time or clearance.

Concentration-time curve. The curve of concentration in blood (or plasma) versus time is a convenient way of describing uptake and disposition of a xenobiotic.

Area under the curve (AUC) is the integral of concentration in blood (plasma) over time. When metabolic saturation and other non-linear processes are absent, AUC is proportional to the absorbed amount of substance.

Biological half-time (or half-life) is the time needed after the end of exposure to reduce the amount in the organism to one-half. As it is often difficult to assess the total amount of a substance, measurements such as the concentration in blood (plasma) are used. The half-time should be used with caution, as it may change, for example, with dose and length of exposure. In addition, many substances have complex decay curves with several half-times.

Bioavailability is the fraction of an administered dose entering the systemic circulation. In the absence of presystemic clearance, or first-pass metabolism, the fraction is one. In oral exposure presystemic clearance may be due to metabolism within the gastrointestinal content, gut wall or liver. First-pass metabolism will reduce the systemic absorption of the substance and instead increase the absorption of metabolites. This may lead to a different toxicity pattern.

Clearance is the volume of blood (plasma) per unit time completely cleared of a substance. To distinguish from renal clearance, for example, the prefix total, metabolic or blood (plasma) is often added.

Intrinsic clearance is the capacity of endogenous enzymes to transform a substance, and is also expressed in volume per unit time. If the intrinsic clearance in an organ is much lower than the blood flow, the metabolism is said to be capacity limited. Conversely, if the intrinsic clearance is much higher than the blood flow, the metabolism is flow limited.

Excretion

Excretion is the exit of a substance and its biotransformation products from the organism.

Excretion in urine and bile. The kidneys are the most important excretory organs. Some substances, especially acids with high molecular weights, are excreted with bile. A fraction of biliary excreted substances may be reabsorbed in the intestines. This process, enterohepatic circulation, is common for conjugated substances following intestinal hydrolysis of the conjugate.

Other routes of excretion. Some substances, such as organic solvents and breakdown products such as acetone, are volatile enough so that a considerable fraction may be excreted by exhalation after inhalation. Small water-soluble molecules as well as fat-soluble ones are readily secreted to the foetus via the placenta, and into milk in mammals. For the mother, lactation can be a quantitatively important excretory pathway for persistent fat-soluble chemicals. The offspring may be secondarily exposed via the mother during pregnancy as well as during lactation. Water-soluble compounds may to some extent be excreted in sweat and saliva. These routes are generally of minor importance. However, as a large volume of saliva is produced and swallowed, saliva excretion may contribute to reabsorption of the compound. Some metals such as mercury are excreted by binding permanently to the sulphydryl groups of the keratin in the hair.

Toxicokinetic models

Mathematical models are important tools to understand and describe the uptake and disposition of foreign substances. Most models are compartmental, that is, the organism is represented by one or more compartments. A compartment is a chemically and physically theoretical volume in which the substance is assumed to distribute homogeneously and instantaneously. Simple models may be expressed as a sum of exponential terms, while more complicated ones require numerical procedures on a computer for their solution. Models may be subdivided in two categories, descriptive and physiological.

In descriptive models, fitting to measured data is performed by changing the numerical values of the model parameters or even the model structure itself. The model structure normally has little to do with the structure of the organism. Advantages of the descriptive approach are that few assumptions are made and that there is no need for additional data. A disadvantage of descriptive models is their limited usefulness for extrapolations.

Physiological models are constructed from physiological, anatomical and other independent data. The model is then refined and validated by comparison with experimental data. An advantage of physiological models is that they can be used for extrapolation purposes. For example, the influence of physical activity on the uptake and disposition of inhaled substances may be predicted from known physiological adjustments in ventilation and cardiac output. A disadvantage of physiological models is that they require a large amount of independent data.

Biotransformation

Biotransformation is a process which leads to a metabolic conversion of foreign compounds (xenobiotics) in the body. The process is often referred to as metabolism of xenobiotics. As a general rule metabolism converts lipid-soluble xenobiotics to large, water- soluble metabolites that can be effectively excreted.

The liver is the main site of biotransformation. All xenobiotics taken up from the intestine are transported to the liver by a single blood vessel (vena porta). If taken up in small quantities a foreign substance may be completely metabolized in the liver before reaching the general circulation and other organs (first pass effect). Inhaled xenobiotics are distributed via the general circulation to the liver. In that case only a fraction of the dose is metabolized in the liver before reaching other organs.

Liver cells contain several enzymes that oxidize xenobiotics. This oxidation generally activates the compound—it becomes more reactive than the parent molecule. In most cases the oxidized metabolite is further metabolized by other enzymes in a second phase. These enzymes conjugate the metabolite with an endogenous substrate, so that the molecule becomes larger and more polar. This facilitates excretion.

Enzymes that metabolize xenobiotics are also present in other organs such as the lungs and kidneys. In these organs they may play specific and qualitatively important roles in the metabolism of certain xenobiotics. Metabolites formed in one organ may be further metabolized in a second organ. Bacteria in the intestine may also participate in biotransformation.

Metabolites of xenobiotics can be excreted by the kidneys or via the bile. They can also be exhaled via the lungs, or bound to endogenous molecules in the body.

The relationship between biotransformation and toxicity is complex. Biotransformation can be seen as a necessary process for survival. It protects the organism against toxicity by preventing accumulation of harmful substances in the body. However, reactive intermediary metabolites may be formed in biotransformation, and these are potentially harmful. This is called metabolic activation. Thus, biotransformation may also induce toxicity. Oxidized, intermediary metabolites that are not conjugated can bind to and damage cellular structures. If, for example, a xenobiotic metabolite binds to DNA, a mutation can be induced (see “Genetic toxicology”). If the biotransformation system is overloaded, a massive destruction of essential proteins or lipid membranes may occur. This can result in cell death (see “Cellular injury and cellular death”).

Metabolism is a word often used interchangeably with biotransformation. It denotes chemical breakdown or synthesis reactions catalyzed by enzymes in the body. Nutrients from food, endogenous compounds, and xenobiotics are all metabolized in the body.

Metabolic activation means that a less reactive compound is converted to a more reactive molecule. This usually occurs during Phase 1 reactions.

Metabolic inactivation means that an active or toxic molecule is converted to a less active metabolite. This usually occurs during Phase 2 reactions. In certain cases an inactivated metabolite might be reactivated, for example by enzymatic cleavage.

Phase 1 reaction refers to the first step in xenobiotic metabolism. It usually means that the compound is oxidized. Oxidation usually makes the compound more water soluble and facilitates further reactions.

Cytochrome P450 enzymes are a group of enzymes that preferentially oxidize xenobiotics in Phase 1 reactions. The different enzymes are specialized for handling specific groups of xenobiotics with certain characteristics. Endogenous molecules are also substrates. Cytochrome P450 enzymes are induced by xenobiotics in a specific fashion. Obtaining induction data on cytochrome P450 can be informative about the nature of previous exposures (see “Genetic determinants of toxic response”).

Phase 2 reaction refers to the second step in xenobiotic meta- bolism. It usually means that the oxidized compound is conjugated with (coupled to) an endogenous molecule. This reaction increases the water solubility further. Many conjugated meta- bolites are actively excreted via the kidneys.

Transferases are a group of enzymes that catalyze Phase 2 reactions. They conjugate xenobiotics with endogenous compounds such as glutathione, amino acids, glucuronic acid or sulphate.

Glutathione is an endogenous molecule, a tripeptide, that is conjugated with xenobiotics in Phase 2 reactions. It is present in all cells (and in liver cells in high concentrations), and usually protects from activated xenobiotics. When glutathione is depleted, toxic reactions between activated xenobiotic metabolites and proteins, lipids or DNA may occur.

Induction means that enzymes involved in biotransformation are increased (in activity or amount) as a response to xenobiotic exposure. In some cases within a few days enzyme activity can be increased several fold. Induction is often balanced so that both Phase 1 and Phase 2 reactions are increased simultaneously. This may lead to a more rapid biotransformation and can explain tolerance. In contrast, unbalanced induction may increase toxicity.

Inhibition of biotransformation can occur if two xenobiotics are metabolized by the same enzyme. The two substrates have to compete, and usually one of the substrates is preferred. In that case the second substrate is not metabolized, or only slowly metabolized. As with induction, inhibition may increase as well as decrease toxicity.

Oxygen activation can be triggered by metabolites of certain xenobiotics. They may auto-oxidize under the production of activated oxygen species. These oxygen-derived species, which include superoxide, hydrogen peroxide and the hydroxyl radical, may damage DNA, lipids and proteins in cells. Oxygen activation is also involved in inflammatory processes.

Genetic variability between individuals is seen in many genes coding for Phase 1 and Phase 2 enzymes. Genetic variability may explain why certain individuals are more susceptible to toxic effects of xenobiotics than others.

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