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Introduction to Toxicology JOHN H. DUFFUS (Fundamental Toxicology, 2nd Ed. 2006; pp. 1 – 3) 1.1 INTRODUCTION Toxicology is the fundamental science of poisons. A poison is generally considered to be any substance that can cause severe injury or death as a result of a physicochemical interaction with living tissue. However, all substances are potential poisons since all of them can cause injury or death following excessive exposure. On the other hand, all chemicals can be used safely if exposure of people or susceptible organisms to chemicals is kept below defined tolerable limits, i.e. if handled with appropriate precautions. If no tolerable limit can be defined, zero exposure methods must be used. Exposure is a function of the amount (or concentration) of the chemical involved, and the time and frequency of its interaction with people or other organisms at risk. For very highly toxic substances, the tolerable exposure may be close to zero. In deciding what constitutes a tolerable exposure, it is essential to have data relating exposure to the production of injury or adverse effect. A problem often arises in deciding what constitutes an injury or adverse effect. An adverse effect is defined as an abnormal, undesirable or harmful change following exposure to the potentially toxic substance. The ultimate adverse effect is death but less severe adverse effects may include altered food consumption, altered body and organ weights, visible pathological changes or simply altered enzyme levels. A statistically significant change from the normal state of the person at risk is not necessarily an adverse effect. The extent of the difference from normal, the consistency of the altered property and the relation of the altered property to the total well-being of the person affected have to be considered. An effect may be considered harmful if it causes functional or anatomical damage, irreversible change in homeostasis or increased susceptibility to other chemical or biological stress, including infectious disease. The degree of harm of the effect can be influenced by the state of health of the organism. Reversible changes may also be harmful, but often they are essentially harmless. An effect which is not harmful is usually reversed when exposure to the potentially toxic chemical ceases. Adaptation of the exposed organism may occur so that it can live normally in spite of an irreversible effect. In immune reactions leading to hypersensitivity or allergic effects, the first exposure to the causative agent may produce no adverse effect, although it sensitizes the organism to respond adversely to future exposures, often at a very low level. The amount of exposure to a chemical required to produce injury varies over a very wide range depending on the chemical and the form in which it occurs. The extent of possible variation in harmful exposure levels is indicated in Table 1.1, which compares median lethal dose (LD50) values for a number of potentially toxic chemicals. The LD50 value is more descriptively called the median lethal dose and is defined below. The LD50 is the statistically derived single dose of a chemical that can be expected to cause death in 50% of a given population of organisms under a defined set of experimental conditions. Where LD50 values are quoted for human beings, they are derived by extrapolation from studies with mammals or from observations following accidental or suicidal exposures. The LD50 has often been used to classify and compare toxicity among chemicals but its value for this purpose is limited. A commonly used classification of this kind is shown in Table 1.2. Such a classification is entirely arbitrary and has some intrinsic weaknesses. For example, it is difficult to see why a substance with an LD50 of 200 mg kg_1 body weight should be regarded only as harmful while one with an LD50 of 199 mg kg_1 body weight is said to be toxic, when the difference in values is minimal. Further, there is no simple relationship between lethality and sublethal toxic effects. In particular, there is no simple relationship between lethality and effects of great concern, such as cancer or abnormal development of the human embryo. Even in relation to lethality, it is not helpful because it gives no measure of the minimum dose that can be lethal and thus no guide to what might be a ‘safe’ exposure level. In decisions relating to chemical safety, the toxicity (hazard) of a substance is less important than the risk associated with its use. Risk is the predicted or actual frequency (probability) of a chemical causing unacceptable harm or effects as a result of exposure of susceptible organisms or ecosystems. Assessment of risk is often assessment of the probability and likely degree of exposure. By comparison with risk, safety is the practical certainty that injury will not result from exposure to a hazard under defined conditions; in other words, the high probability that injury will not result. Practical certainty is defined as a numerically specified low risk or socially acceptable risk applied in decision making for risk management. In assessing permissible exposure conditions for chemicals, uncertainty factors are applied. A threshold of exposure above which an adverse effect can occur (and below which no such effect is observed) is defined from the available data, and this is divided by an uncertainty factor to lower it to a value that regulatory toxicologists can regard as safe beyond doubt. An uncertainty factor may be defined as a mathematical expression of uncertainty that is used to protect populations from hazards that cannot be assessed with high precision. For example, the 1977 report of the US National Academy of Sciences Safe Drinking Water Committee proposed the following guidelines for selecting uncertainty (safety) factors to be used in conjunction with no observed effect level (NOEL) data. The NOEL should be divided by the following uncertainty factors: 1. An uncertainty factor of 10 should be used when valid human data based on chronic exposure are available. 2. An uncertainty factor of 100 should be used when human data are inconclusive, e.g. limited to acute exposure histories, or absent, but when reliable animal data are available for one or more species. 3. An uncertainty factor of 1000 should be used when no long-term, or acute human data are available and experimental animal data are scanty. This approach is subjective and is being continually updated. Safety control often involves the assessment of ‘acceptable’ risk since total elimination of risk is often impossible. ‘Acceptable’ risk is the probability of suffering disease or injury that will be tolerated by an individual, group, or society. Assessment of risk depends on scientific data but its ‘acceptability’ is influenced by social, economic and political factors, and by the perceived benefits arising from a chemical or process. INTRODUCTION TO TOXICOLOGY (Cassaret and Doull’s) Toxicology is the study of the adverse effects of chemical or physical agents on living organisms. A toxicologist is trained to examine and communicate the nature of those effects on human, animal, and environmental health. Toxicological research examines the cellular, biochemical, and molecular mechanisms of action as well as functional effects such as neurobehavioral and immunological, and assesses the probability of their occurrence. Fundamental to this process is characterizing the relation of exposure (or dose) to the response. Risk assessment is the quantitative estimate of the potential effects on human health and environmental significance of various types of chemical exposures (e.g., pesticide residues on food, contaminants in drinking water). The variety of potential adverse effects and the diversity of chemicals in the environment make toxicology a broad science, which often demands specialization in one area of toxicology. Our society’s dependence on chemicals and the need to assess potential hazards have made toxicologists an increasingly important part of the decision-making processes. Different Areas of Toxicology The professional activities of toxicologists fall into three main categories: descriptive, mechanistic, and regulatory (Fig. 2-1). Although each has distinctive characteristics, each contributes to the other, and all are vitally important to chemical risk assessment (see Chap. 4). A mechanistic toxicologist is concerned with identifying and understanding the cellular, biochemical, and molecular mechanisms by which chemicals exert toxic effects on living organisms (see Chap. 3 for a detailed discussion of mechanisms of toxicity). The results of mechanistic studies are very important in many areas of applied toxicology. In risk assessment, mechanistic data may be very useful in demonstrating that an adverse outcome (e.g., cancer, birth defects) observed in laboratory animals is directly relevant to humans. For example, the relative toxic potential of organophosphate insecticides in humans, rodents, and insects can be accurately predicted on the basis of an understanding of common mechanisms (inhibition of acetylcholinesterase) and differences in biotransformation for these insecticides among the different species. Similarly, mechanistic data may be very useful in identifying adverse responses in experimental animals that may not be relevant to humans. For example, the propensity of the widely used artificial sweetener saccharin to cause bladder cancer in rats may not be relevant to humans at normal dietary intake rates. This is because mechanistic studies have demonstrated that bladder cancer is induced only under conditions where saccharin is at such a high concentration in the urine that it forms a crystalline precipitate (Cohen, 1998). Dose–response studies suggest that such high concentrations would not be achieved in the human bladder even after extensive dietary consumption. Mechanistic data are also useful in the design and production of safer alternative chemicals and in rational therapy for chemical poisoning and treatment of disease. For example, the drug thalidomide was originally marketed in Europe and Australia as a sedative agent for pregnantwomen. However, itwas banned for clinical use in 1962 because of devastating birth defects that occurred if the drug was ingested during a critical period in pregnancy. But mechanistic studies over the past several decades have demonstrated that this drug may have a unique molecular mechanism of action that interferes with the expression of certain genes responsible for blood vessel formation (angiogenesis). With an understanding of this mechanism, thalidomide has been “rediscovered” as a valuable therapeutic agent that may be highly effective in the treatment of certain infectious diseases (e.g., leprosy and AIDS), a variety of inflammatory diseases, and some types of cancer. This provides an interesting example of how a highly toxic drug with selectivity toward a specific population (pregnant women) can be used safely with proper precautions. Following its approval for therapeutic use in 1998, a program was established that required all clinicians, pharmacists, and patients that receive thalidomide to enroll in a specific program (System for Thalidomide Education and Prescribing Safety, STEPS). The population at risk for the potential teratogenic effects of thalidomide (all women of childbearing age) were required to use two forms of birth control, and also have a negative pregnancy test within 24 hours of beginning therapy, and periodically the patients registered with the STEPS program, 6000 were females of childbearing age. Remarkably, after 6 years of use, only one patient actually received thalidomide during her pregnancy. She initially tested negative at the beginning of therapy; on a subsequent test she was identified as positive, and the drug was stopped. The pregnancy ended up as a miscarriage (Uhl et al., 2006). Thus, a clear understanding of mechanism of action led to the development of strict prescribing guidelines and patient monitoring, thereby allowing a potentially dangerous drug to be used safely and effectively to treat disease in tens of thousands of patients who would otherwise not have benefited from the therapeutic actions of the drug (Lary et al., 1999). In addition to aiding directly in the identification, treatment, and prevention of chemical toxicity, an understanding of the mechanisms of toxic action contributes to the knowledge of basic physiology, pharmacology, cell biology, and biochemistry. The advent of new technologies in molecular biology and genomics now provide mechanistic toxicologists with the tools to explore exactly how humans may differ from laboratory animals in their response to toxic substances. These same tools are also being utilized to identify individuals who are genetically susceptible to factors in the environment or respond differently to a chemical exposure. For example, it is nowrecognized that a small percentage of the population genetically lacks the ability to detoxify the chemotherapeutic drug, 6-mercaptopurine, used in the treatment of some forms of leukemia. Young children with leukemia who are homozygous for this genetic trait (about one in 300) may experience serious toxic effects from a standard therapeutic dose of this drug. Numerous genetic tests for polymorphisms in drug metabolizing enzymes and transporters are now available that can identify genetically susceptible individuals in advance of pharmacological treatment (Eichelbaum et al., 2006). These new areas of “pharmacogenomics” and “toxicogenomics” provides an exciting opportunity in the future for mechanistic toxicologists to identify and protect genetically susceptible individuals from harmful environmental exposures, and to customize drug therapies that enhance efficacy and minimize toxicity, based on an individual’s genetic makeup. A descriptive toxicologist is concerned directly with toxicity testing, which provides information for safety evaluation and regulatory requirements. The appropriate toxicity tests (as described later in this chapter and other chapters) in cell culture systems or experimental animals are designed to yield information to evaluate risks posed to humans and the environment from exposure to specific chemicals. The concern may be limited to effects on humans, as in the case of drugs and food additives. Toxicologists in the chemical industry, however, must be concerned not only with the risk posed by a company’s chemicals (insecticides, herbicides, solvents, etc.) to humans but also with potential effects on fish, birds, and plants, as well as other factors that might disturb the balance of the ecosystem. Descriptive toxicology is of course not divorced from mechanistic studies, as such studies provide important clues to a chemical’s mechanism of action, and thus contribute to the development of mechanistic toxicology through hypothesis generation. Such studies are also a key component of risk assessments that are used by regulatory toxicologists. The recent advent of so-called “omics” technologies (genomics, transcriptomics, proteomics, metabonomics, etc.) form the basis of the emerging subdiscipline of toxicogenomics. The application of these new technologies to toxicity testing is in many ways “descriptive” in nature, yet affords great mechanistic insights into how chemicals produce their toxic effects. This exciting new area of toxicology is discussed in more detail later in the chapter. A regulatory toxicologist has the responsibility for deciding, on the basis of data provided by descriptive and mechanistic toxicologists, whether a drug or other chemical poses a sufficiently low risk to be marketed for a stated purpose or subsequent human or environmental exposure resulting from its use. The Food and Drug Administration (FDA) is responsible for allowing drugs, cosmetics, and food additives to be sold in the market according to the Federal Food, Drug and Cosmetic Act (FFDCA). The U.S. Environmental Protection Agency (EPA) is responsible for regulating most other chemicals according to the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), the Toxic Substances Control Act (TSCA), the Resource Conservation and Recovery Act (RCRA), the Safe Drinking Water Act, and the Clean Air Act. In 1996, the U.S. Congress passed the Food Quality Protection Act (FQPA) which fundamentally changed the pesticide and food safety laws under FIFRA and FFDCA requiring stricter safety standards particularly for infants and children, who were recognized as more susceptible to health effects of pesticides. The EPA is also responsible for enforcing the Comprehensive Environmental Response, Compensation and Liability Act [CERCLA, later revised as the Superfund Amendments Reauthorization Act (SARA)], more commonly called the Superfund Act. This regulation provides direction and financial support for the cleanup of waste sites that contain toxic chemicals that may present a risk to human health or the environment. The Occupational Safety and Health Administration (OSHA) of the Department of Labor was established to ensure that safe and healthful conditions exist in the workplace. The National Institute for Occupational Safety and Health (NIOSH) as part of the Centers for Disease Control and Prevention (CDC) in the Department of Health and Human Services is responsible for conducting research and making recommendations for the prevention of work-related injury and illness. The Consumer Product Safety Commission is responsible for protecting consumers from hazardous household substances, whereas the Department of Transportation (DOT) ensures that materials shipped in interstate commerce are labeled and packaged in a manner consistent with the degree of hazard they present. Regulatory toxicologists are also involved in the establishment of standards for the amount of chemicals permitted in ambient air, industrial atmospheres, and drinking water, often integrating scientific information from basic descriptive and mechanistic toxicology studies with the principles and approaches used for risk assessment (see Chap. 4). In addition to the above categories, there are other specialized areas of toxicology such as forensic, clinical, and environmental toxicology. Forensic toxicology is a hybrid of analytic chemistry and fundamental toxicological principles. It is concerned primarily with the medicolegal aspects of the harmful effects of chemicals on humans and animals. The expertise of forensic toxicologists is invoked primarily to aid in establishing the cause of death and determining its circumstances in a postmortem investigation (see Chap. 31). Clinical toxicology designates an area of professional emphasis in the realm of medical science that is concerned with disease caused by or uniquely associated with toxic substances (see Chap. 32). Generally, clinical toxicologists are physicians who receive specialized training in emergency medicine and poison management. Efforts are directed at treating patients poisoned with drugs or other chemicals and at the development of new techniques to treat those intoxications. Public contact about treatment and prevention is often through the national network of poison control centers. Environmental toxicology focuses on the impacts of chemical pollutants in the environment on biological organisms. Although toxicologists concerned with the effects of environmental pollutants on human health fit into this definition, it is most commonly associated with studies on the impacts of chemicals on nonhuman organisms such as fish, birds, terrestrial animals, and plants. Ecotoxicology is a specialized area within environmental toxicology that focuses more specifically on the impacts of toxic substances on population dynamics in an ecosystem. The transport, fate, and interactions of chemicals in the environment constitute a critical component of both environmental toxicology and ecotoxicology. CLASSIFICATION OF TOXIC AGENTS (Cassaret and Doull’s) Toxic agents are classified in a variety of ways, depending on the interests and needs of the classifier. In this textbook, for example, toxic agents are discussed in terms of their target organs (liver, kidney, hematopoietic system, etc.), use (pesticide, solvent, food additive, etc.), source (animal and plant toxins), and effects (cancer, mutation, liver injury, etc.). The term toxin generally refers to toxic substances that are produced by biological systems such as plants, animals, fungi, or bacteria. The term toxicant is used in speaking of toxic substances that are produced by or are a by-product of anthropogenic (human-made) activities. Thus, zeralanone, produced by a mold, is a toxin, whereas “dioxin” [2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)], produced during the production and/or combustion of certain chlorinated organic chemicals, is a toxicant. Some toxicants can be produced by both natural and anthropogenic activities. For example, polyaromatic hydrocarbons are produced by the combustion of organic matter, which may occur both through natural processes (e.g., forest fires) and through anthropogenic activities (e.g., combustion of coal for energy production; cigarette smoking). Arsenic, a toxic metalloid, may occur as a natural contaminant of groundwater or may contaminate groundwater secondary to industrial activities. Generally, such toxic substances are referred to as toxicants, rather than toxins, because, although they are naturally produced, they are not produced by biological systems. Toxic agents may also be classified in terms of their physical state (gas, dust, liquid), their chemical stability or reactivity (explosive, flammable, oxidizer), general chemical structure (aromatic amine, halogenated hydrocarbon, etc.), or poisoning potential (extremely toxic, very toxic, slightly toxic, etc.). Classification of toxic agents on the basis of their biochemical mechanisms of action (e.g., alkylating agent, cholinesterase inhibitor, methemoglobin producer) is usually more informative than classification by general terms such as irritants and corrosives. But more general classifications such as air pollutants, occupation-related agents, and acute and chronic poisons can provide a useful focus on a specific problem. It is evident from this discussion that no single classification is applicable to the entire spectrum of toxic agents and that combinations of classification systems or a classification based on other factors may be needed to provide the best rating system for a special purpose. Nevertheless, classification systems that take into consideration both the chemical and the biological properties of an agent and the exposure characteristics are most likely to be useful for regulatory or control purposes and for toxicology in general. CHARACTERISTICS OF EXPOSURE (Cassaret and Doull’s) Toxic effects in a biological system are not produced by a chemical agent unless that agent or its metabolic breakdown (biotransformation) products reach appropriate sites in the body at a concentration and for a length of time sufficient to produce a toxic manifestation. Many chemicals are of relatively low toxicity in the “native” form but, when acted on by enzymes in the body, are converted to intermediate forms that interfere with normal cellular biochemistry and physiology. Thus, whether a toxic response occurs is dependent on the chemical and physical properties of the agent, the exposure situation, how the agent is metabolized by the system, the concentration of the active form at the particular target site(s), and the overall susceptibility of the biological system or subject. Thus, to characterize fully the potential hazard of a specific chemical agent, we need to know not only what type of effect it produces and the dose required to produce that effect but also information about the agent, the exposure, and its disposition by the subject. Two major factors that influence toxicity as it relates to the exposure situation for a specific chemical are the route of exposure, and the duration, and frequency of exposure. Route and Site of Exposure The major routes (pathways) by which toxic agents gain access to the body are the gastrointestinal tract (ingestion), lungs (inhalation), skin (topical, percutaneous, or dermal), and other parenteral (other than intestinal canal) routes. Toxic agents generally produce the greatest effect and the most rapid response when given directly into the bloodstream (the intravenous route). An approximate descending order of effectiveness for the other routes would be inhalation, intraperitoneal, subcutaneous, intramuscular, intradermal, oral, and dermal. The “vehicle” (the material in which the chemical is dissolved) and other formulation factors can markedly alter absorption after ingestion, inhalation, or topical exposure. In addition, the route of administration can influence the toxicity of agents. For example, an agent that acts on the CNS, but is efficiently detoxified in the liver, would be expected to be less toxic when given orally than when given via inhalation, because the oral route requires that nearly all of the dose pass through the liver before reaching the systemic circulation and then the CNS. Occupational exposure to toxic agents most frequently results from breathing contaminated air (inhalation) and/or direct and prolonged contact of the skin with the substance (dermal exposure), whereas accidental and suicidal poisoning occurs most frequently by oral ingestion. Comparison of the lethal dose of a toxic substance by different routes of exposure often provides useful information about its extent of absorption. In instances when the toxic dose after oral or dermal administration is similar to the toxic dose after intravenous administration, the assumption is that the toxic agent is absorbed readily and rapidly. Conversely, in cases where the toxic dose by the dermal route is several orders of magnitude higher than the oral toxic dose, it is likely that the skin provides an effective barrier to absorption of the agent. Toxic effects by any route of exposure can also be influenced by the concentration of the agent in its vehicle, the total volume of the vehicle and the properties of the vehicle to which the biological system is exposed, and the rate at which exposure occurs. Studies in which the concentration of a chemical in the blood is determined at various times after exposure are often needed to clarify the role of these and other factors in the toxicity of a compound. For more details on the absorption of toxicants. Duration and Frequency of Exposure Toxicologists usually divide the exposure of experimental animals to chemicals into four categories: acute, subacute, subchronic, and chronic. Acute exposure is defined as exposure to a chemical for less than 24 hours, and examples of exposure routes are intraperitoneal, intravenous, and subcutaneous injection; oral intubation; and dermal application. Whereas acute exposure usually refers to a single administration, repeated exposures may be given within a 24-hours period for some slightly toxic or practically nontoxic chemicals. Acute exposure by inhalation refers to continuous exposure for less than 24 hours, most frequently for 4 hours. Repeated exposure is divided into three categories: subacute, subchronic, and chronic. Subacute exposure refers to repeated exposure to a chemical for 1 month or less, subchronic for 1 to 3 months, and chronic for more than 3 months, although usually this refers to studies with at least 1 year of repeated dosing. These three categories of repeated exposure can be by any route, but most often they occur by the oral route, with the chemical added directly to the diet. In human exposure situations, the frequency and duration of exposure are usually not as clearly defined as in controlled animal studies, but many of the same terms are used to describe general exposure situations. Thus,workplace or environmental exposures may be described as acute (occurring from a single incident or episode), subchronic (occurring repeatedly over several weeks or months), or chronic (occurring repeatedly for many months or years). For many chemicals, the toxic effects that follow a single exposure are quite different from those produced by repeated exposure. For example, the primary acute toxic manifestation of benzene is central nervous system (CNS) depression, but repeated exposures can result in bone marrow toxicity and an increased risk for leukemia. Acute exposure to chemicals that are rapidly absorbed is likely to produce immediate toxic effects but also can produce delayed toxicity that may or may not be similar to the toxic effects of chronic exposure. Conversely, chronic exposure to a toxic chemical may produce some immediate (acute) effects after each administration in addition to the long-term, low-level, or chronic effects of the toxic substance. In characterizing the toxicity of a specific chemical, it is evident that information is needed not only for the singledose (acute) and long-term (chronic) effects but also for exposures of intermediate duration. The other time-related factor that is important in the temporal characterization of repeated exposures is the frequency of exposure. The relationship between elimination rate and frequency of exposure is shown in Fig. 2-2. A chemical that produces severe effects with a single dose may have no effect if the same total dose is given in several intervals. For the chemical depicted by line B in Fig. 2-2, in which the half-life for elimination (time necessary for 50% of the chemical to be removed from the bloodstream) is approximately equal to the dosing frequency, a theoretical toxic concentration (shown conceptually as two Concentration Units in Fig. 2-2) is not reached until the fourth dose, whereas that concentration is reached with only two doses for chemical A, which has an elimination rate much slower than the dosing interval (time between each repeated dose). Conversely, for chemical C, where the elimination rate is much shorter than the dosing interval, a toxic concentration at the site of toxic effect will never be reached regardless of how many doses are administered. Of course, it is possible that residual cell or tissue damage occurs with each dose even though the chemical itself is not accumulating. The important consideration, then, is whether the interval between doses is sufficient to allow for complete repair of tissue damage. It is evident that with any type of repeated exposure, the production of a toxic effect is influenced not only by the frequency of exposure but may, in fact, be totally dependent on the frequency rather than the duration of exposure. Chronic toxic effects may occur, therefore, if the chemical accumulates in the biological system (rate of absorption exceeds the rate of biotransformation and/or excretion), if it produces irreversible toxic effects, or if there is insufficient time for the system to recover from the toxic damage within the exposure frequency interval.