An Introduction to Interdisciplinary Toxicology: From Molecules to Man

An Introduction to Interdisciplinary Toxicology: From Molecules to Man integrates the various aspects of toxicology, from “simple molecular systems, to complex human communities, with expertise from a spectrum of interacting disciplines. Chapters are written by specialists within a given subject, such as a chemical engineer, nutritional scientist, or a microbiologist, so subjects are clearly explained and discussed within the toxicology context. Many chapters are comparative across species so that students in ecotoxicology learn mammalian toxicology and vice versa. Specific citations, further reading, study questions, and other learning features are also included.

The book allows students to concurrently learn concepts in both biomedical and environmental toxicology fields, thus better equipping them for the many career opportunities toxicology provides. This book will also be useful to those wishing to reference how disciplines interact within the broad field of toxicology.

• Covers major topics and newer areas in toxicology, including nanotoxicology, Tox21, epigenetic toxicology, and organ-specific toxicity
• Includes a variety of perspectives to give a complete understanding of toxicology
• Written by specialists within each subject area, e.g., a chemical engineer, to ensure concepts are clearly explained

• Language: English
• Publisher: Academic Press
• Release date: Feb 18, 2020
• ISBN: 9780128136034

Book preview

toxicology.

Preface

Carey N. Pope and Jing Liu

The Interdisciplinary Toxicology Program (ITP) was established at Oklahoma State University (OSU) in 2012, with the recognition that complex environmental issues of our time surrounding chemical contamination will require the efforts of investigators across disciplines and the cross-training of their students to be effective investigators. Faculty and students in our program come from 12 different departments, 6 colleges, and 2 campuses. Our earlier experience with an undergraduate toxicology program at the University of Louisiana at Monroe emphasized the value of starting simple in developing and transferring knowledge in toxicology through coursework and laboratory experiences, highlighting important concepts and skills in easy-to-understand approaches. This same concept of education and training applies to graduate students in an interdisciplinary program, with students coming from diverse multiple disciplines and sometimes very different experiences.

This book is modeled after one of the courses in the OSU ITP, Toxicology: from molecules to ecosystems. The course begins with principles and goes on to cover from toxicant-target interactions to proteotoxicity, cellular responses, toxicokinetics, organ systems, ecotoxicology, forensics, population effects, the sociology of chemical contamination episodes, and other topics, matching the strengths of the participating faculty and the interests of their students. While covering the subject matter can be a challenge for both the students and the instructors, most agree that synergy can develop when bringing different emphasis areas, concepts, and approaches together. Active participation between the students and instructors is an important part of the course and facilitates an understanding among all for their specific interests and experiences.

One advantage for putting this book together was a necessary emphasis on what we were teaching and how it could be made more succinct and clear, in addition to having the opportunity to recruit other OSU faculty for coverage of new areas of emphasis. Expert authors from other institutions contributed chapters as well, and a number of those have already visited or will visit OSU as part of our annual ITP symposium. We are indebted to the efforts of all of the chapter contributors without which completion of the book could not have happened. We hope that our book provides an easy-to-understand survey of timely topics in toxicology suitable for graduate students across disciplines entering into this exciting area of investigation.

September 2019

Part I

General concepts

Outline

Chapter 1 History and basic concepts of toxicology

Chapter 2 Absorption, distribution, and excretion in complex organisms

Chapter 3 Xenobiotic metabolism and disposition

Chapter 1

History and basic concepts of toxicology

Carey N. Pope¹, Daniel Schlenk² and Frédéric J. Baud³,⁴,⁵,    ¹Department of Physiological Sciences, College of Veterinary Medicine, Interdisciplinary Toxicology Program, Oklahoma State University, Stillwater, OK, United States,    ²Department of Environmental Sciences, University of California, Riverside, CA, United States,    ³Medical and Toxicological Critical Care Department, Assistance Publique—Hôpitaux de Paris, Necker Hospital, Paris, France,    ⁴University Paris Diderot, Paris, France,    ⁵EA7323 Evaluation of therapeutics and pharmacology in perinatality and pediatrics—University Hospital Cochin—Broca—Hôtel Dieu, Site Tarnier, University Paris Descartes, Paris, France

Abstract

Toxicology is the study of adverse effects of chemical substances on living organisms. The Society of Toxicology more broadly defined toxicology as the study of adverse effects of chemical, physical, or biological agents on living organisms and the ecosystem, including the prevention and ameliorization of such adverse effects. This definition still leaves some loose ends, for example, a toxicologist could study the macromolecular interactions of a toxicant and the receptor with which it binds to elicit a toxic response, without evaluating an intact living organism or system. On the other end of the spectrum, a toxicologist can study the effects of a contaminant on an entire ecosystem with multiple interacting species across the taxonomic spectrum.

Key to any definition of toxicology however is the word adverse. An adverse effect can be defined as a response to a substance which is harmful or unfavorable. Thus toxicologists study agents which elicit harmful or unfavorable effects on living systems. While some substances can elicit undeniably adverse effects under certain conditions, those same substances may elicit no change or some change which is difficult to be defined clearly as being harmful under other conditions. The wide range of substances eliciting toxic responses can be classified as either toxicants (naturally occurring or synthetic xenobiotics) or toxins (substances produced by other living organisms that elicit toxic responses). But as will be noted later, all substances have the potential to elicit adverse effects.

The overlying concept of this book is an introduction to toxicology from multiple levels of organization and varying perspectives. Multiple emphasis areas fit under the umbrella of toxicology. For example, molecular toxicology pertains to the interaction between toxic substances and biomolecules such as nucleic acids, proteins, and lipids, and the adverse consequences which follow. Cellular toxicology focuses on how cells, as the basic unit of living organisms, are affected by and respond to toxicant exposure. Organ system toxicology studies how the functions of various organs within more complex species are affected, while organismal toxicology considers the overall responses exhibited by the intact organism. Ecotoxicology studies how substances affect multiple levels of organization from the molecular through the organismal level, and the subsequent impacts on populations and/or communities within an ecosystem. Other subdisciplines of toxicology such as nanotoxicology, veterinary toxicology, clinical toxicology, and others have their own selective emphases and are discussed in later chapters.

Keywords

Dose–response; interactions; time dependent; tolerance; history; subdisciplines

1.1 A brief history of toxicology

There is substantial evidence indicating that humans have been aware of, and in some cases utilized, the toxicity of various substances since antiquity. While there is little evidence of poisonings in the Paleolithic and Neolithic periods in Europe, around 18,000 years ago Maasai hunters in Kenya used arrow and dart poisons (likely cardiac glycosides of Strophanthus species) to increase the effectiveness of their weapons. Indeed the term toxicology is derived from the Greek terms toxikos (bow) and toxicon (poison into which arrowheads are dipped).¹

In the bronze (3000–1000 years BCE) and iron ages (800–100 years BCE), people started to communicate with writing, providing lasting documentation of accidental and intentional intoxications and the use of toxic substances in executions. During the Bronze Age, metal alloys were first developed using tin, aluminum, lead, manganese, and other trace elements. During the Iron Age, the development of iron and steel industries was instrumental in the maintenance of power and order by European monarchies and feudal overlords. One can assume that human exposure to heavy metals was a constant threat due to the smelting, iron casting, and other activities such as painting and tanning.

In the past, medical toxicology concerned natural substances including metals, plants, fungi such as mushrooms and mycotoxins (ergotism), bacterial exotoxin (botulism), and venomous animals as well as carbon oxides produced by combustion of carbonaceous materials. The Eber’s papyrus, an ancient Egyptian text written around 1500 BCE, is among the earliest of medical texts, describing a variety of ancient poisons including aconite, antimony, arsenic, cyanogenic glycosides, hemlock, lead, mandrake, opium, and wormwood.

The basis of pharmacology was clearly stated in Phaedo by Plato (428–348 BCE), and further developed by Aristotle (384–322 BCE). At this time, the toxicity of plants and venomous animals was well known as illustrated by the modus operandi for Socrates’ sanctioned execution by self-ingestion of hemlock (470–399 BCE), while much later the Egyptian queen Cleopatra died from a self-inflicted fatal snake bite (51–30 BCE). The Roman empire followed by the Middle Age and Renaissance inaugurated a long period during which murder using poisonous substances was a common practice, using knowledge held by wizards and alchemists. The Greek physician Galen (c. CE 129–200) described Mithridates’ experiences in a series of books on Antidotes. Chemical warfare and infectious agents were commonly used during sieges. A number of historians suggested a relationship between the large use of lead for the numerous pipelines supplying Rome’s drinking water and chronic lead poisoning of the Roman population leading to the twilight and eventual fall of the Roman Empire in the mid-5th century CE.

The bean of the Calabar plant (Physostigma venonosum) and seeds of a variety of other plants were used in Africa and Madagascar for likely hundreds of years as ordeal poisons to determine guilt of someone accused of a crime. While the substance and methods for using an ordeal poison varied, the suspect was typically forced to eat or drink the substance and the reaction was observed. If the material was expelled by vomiting, he or she was assumed to be innocent. If the individual did not eliminate the poison, toxicity would follow shortly and the accused would be considered guilty by the negative outcome.¹,²

The term poison appeared first in the English literature around CE 1225 to describe a potion that was prepared with deadly ingredients. Since the Middle Age, members of aristocracy used tasters to shield themselves from potential poisoners by having them first sample their beverages and meals before consuming themselves. Interestingly, the concept of making a toast arose from a common fear of poisoning. It was believed that if all present would drink from the same container at the same time, it would likely be devoid of any deadly poison. Obviously, a martyr (person who will die for a cause) could make this strategy less protective.

During the Italian Renaissance, Paracelsus (1493–1541) at the University of Ferrara in Italy described a number of principles of human toxicology (see Fig. 1.1). The most well known is the prominent role of the dose of the substance in toxicity, reported as No substances are safe, all substances are poisonous. The major parameter of toxicity is the dose. However, Paracelsus’ ideology should not be restricted to this major principle. His work led to the description of some types of toxicants as xenobiotics (toxic substances originating from outside of the human body) and to the field of organ toxicology.

Figure 1.1 Commemorative to Paracelsus, University of Ferrara, Italy.

In this University, the great scientist Theophrastus Bombastus von Hohenheim Paracelsus obtained a degree in Medicine. Initiator of a new system in therapeutics. Master of the modern medical sciences. Naturalist philosopher of Europe. Pioneer of Toxicology.

In the mid-17th century, Bernardino Ramazzini (1633–1714) first developed the area of occupational medicine. In 1700 he wrote De Morbis Artificum Diatriba (diseases of workers), the first comprehensive text discussing the relationship between disease and workplace hazards. Ramazzini described diseases associated with 54 occupations, including solvent poisoning in painters, mercury poisoning in mirror makers, and pulmonary diseases in miners. Around 1775, Sir Percivall Pott uncovered the association between workplace exposures and cancer, when he reported a high incidence of scrotal cancer in English chimney sweeps, whose occupation was associated with direct and chronic exposure to incomplete combustion products such as complex polycyclic aromatic hydrocarbons.

About one century later, the French physician Bonaventure Orfila (1787–1853) highlighted the role of toxicology as a distinct discipline separated from clinical medicine and pharmacology. His treatise Traité des Poisons (1814) is regarded as the foundation of experimental and forensic toxicology, promoting the use of chemical analysis and autopsy for medicolegal purposes. The French physician Claude Bernard (1813–78) was instrumental in discovering the mechanism of toxicity of carbon monoxide through its binding to hemoglobin. He also provided the first compelling evidence for a synapse between a motor neuron and the muscle cell with which it communicates. Interestingly, much of Bernard’s work in this context relied on the effects of one of the arrow poisons, curare. He promoted experimental studies in physiology to assess the accuracy of hypotheses regarding mechanism of toxicity and advised the use of poisons to study organ function, summarized in his aphorism: "The poison is for the physiologist like the scalpel is for the surgeon."

While one can identify through literature when chemicals were first being used for poisonings, it is more difficult to determine a time when people first started using substances for recreational purposes. It is known however that marijuana (Cannabis sp.) has been used for millennia. Many natural plants, herbs, and seeds contain psychoactive substances which have been used in traditional medicines. Written communication did not start in China until the 1700s, but it is suggested that the Chinese have been using herbal medicines for likely thousands of years. In Europe in the 16th century, Paracelsus was promoting the medical use of opium. In the 17th century, the English physician Thomas Sydenham proposed a formulation of opium tincture for various purposes.

Alice Hamilton (1869–1970) was first to highlight occupational toxicology. By living and working in a working class neighborhood in Chicago, she identified dangerous trades including those working with rubber, dyes, lead, enamelware, copper, mercury, and explosives, documenting the different types of disorders. Her work on lead intoxication was one of the first that focused on gender differences in response to toxicants.

The awareness of toxicological hazards to which the general population may be exposed is a relatively recent phenomenon. The establishment of regulatory authorities appeared only very recently. Interestingly, in France, a progressive and continuing decrease in attempted murders using poisonous substances was associated with increasing legal freedom to divorce starting in the late 18th century. The US Pure Food and Drug Act of 1906 was the first federal legislative antipoisoning regulatory initiative.¹ The Federal Caustic Poison Act of 1927 was the first federal legislation to specifically address household poisonings. In fact, the US Food and Drug Administration was born out of a major drug-related poisoning disaster. In the early–mid 1930s, sulfamides were developed as potent antimicrobial agents. Unfortunately, the antimicrobials were given intravenously in a diethylene glycol solvent, leading to the deaths of hundreds of patients from acute renal failure. After this tragedy, the policies that required safety testing of new drugs before marketing were developed and implemented. Nowadays, in addition to therapeutics and drugs of abuse, environmental contaminants, and ecotoxicology are major concerns, and governmental agencies are addressing to change large-scale activities. The development of Poison Control Centers in the mid-20th century was also a major step worldwide for vigilant tracking of human responses to xenobiotics, determining toxic relationships between exposure to newly released or currently marketed drugs and environmental contaminants.

1.2 Important concepts in toxicology

Chemical contamination episodes occur relatively often and can be found in reports by various news outlets. The public’s perception of these events plays a major role in how communities deal with such episodes and how those communities, interest groups, and local, state, federal, and international governments may respond. A basic understanding of the principles of toxicology is important for communicating the relative nature of chemical hazards and informing public perception.

1.2.1 The dose–response relationship

A key factor for placing in context any intoxication or chemical contamination event, and a hallmark of toxicology as a scientific field, is the concept of the dose–response relationship, that is, the relationship between the incidence or magnitude of a toxic response and the extent of the chemical exposure. As noted in Section 1.1, the Swiss physician Theophrastus von Hohenheim (1493–1541), who took the name Paracelsus later in life, was an early proponent of the application of chemistry in medicine and medical education.³ In the 16th century, Paracelsus was the first to propose that a predictable relationship exists between the extent of exposure to a substance and its relative therapeutic or toxic effect. His quote dosis sola facit venenum (dose alone makes the poison) is widely paraphrased. Because of the paramount importance of the dose–response relationship in chemical toxicity, Paracelsus is commonly recognized as the father of toxicology.⁴

Toxicity can be defined as the inherent capacity of a chemical to do harm to a living organism. Hazard is defined as the probability or practical certainty that an adverse effect (harm) will occur when a chemical is used under stated conditions (amount, dose, concentration, exposure, duration of exposure, use of personal protective equipment, etc.). In contrast, safety is the practical certainty that toxicity will not occur when a chemical is used under defined conditions. The hazard/safety associated with the use of any chemical therefore depends not only on its inherent chemical properties, but also on the likelihood (and if so the extent) of exposure when the chemical is used under defined conditions. An important corollary of Paracelsus’ centuries-old concept is that while all chemicals can elicit toxicity, any chemical can be used safely if its toxic potential is recognized and the exposures are effectively controlled.

Exposures can be considered in a number of ways. They can be based on the amount of chemical in the ambient environment, on the amount of chemical absorbed into the organism, or most importantly on the amount of chemical that reaches receptors within an organism that initiate a toxic response. While it is appreciated that the magnitude of a toxic response is related to the concentration or dose of the toxicant, what is critical is the concentration of the chemical at the receptor site, with the toxicant–receptor interaction constituting a molecular initiating event that progresses through key events to an ultimate toxic response. In essence, a toxicant must interact with a receptor on/in a cell or tissue to initiate toxicity. Theoretical and practical implications of the toxicant–receptor interactions continue to impact how chemicals are evaluated and regulated for protecting public health and the environment.⁴ The frequency and duration, when repeated exposures occur, are also vital in the expression of dose-related toxicity.

All chemicals have the capacity to elicit toxic responses. It is therefore important to consider a chemical’s toxicity in context with other substances. The most recognized endpoint in toxicology for comparing substances is historically the lethal dose 50 (LD50), that is, a statistically determined dose of a chemical that leads to death in 50% of a group/population of exposed organisms. The standard LD50 approach has been progressively replaced in many areas by assessment with other methods such as estimating maximum tolerated dose (MTD) approaches generally requiring less animals to derive an estimate of acute lethality.

In ecological studies, the environmental medium is typically used for exposure, with those exposures being quantified by the substance concentration within the medium. Thus toxicity is often expressed as the concentration in the medium that kills 50% of the exposed population, that is, the LC50. It is important to differentiate between concentration and dose, since the former does not measure internal (target/receptor site) content of the chemical but only measures the chemical’s concentration in the medium. Concentration is also generally used to characterize in vitro and other exposures, for example, in inhalation toxicity studies.

Knowledge of doses or concentrations of a chemical that either do or do not elicit toxicity is essential in characterizing that chemical’s relative potency. There are two major types of dose–response or concentration–response relationships, that is, those which exhibit a threshold and those which do not. Fig. 1.2 provides examples of both (data in these figures are not from any real study but are merely for example purposes). In Fig. 1.2A, both chemical X and chemical Y elicit a dose-related increase in toxicity. With lower exposures (0.03 mg/kg/day for chemical X and 0.03−1 mg/kg/day for chemical Y), no incidence of the response is noted. As the dose increases, however, the percent of individuals showing toxicity also increases. Note that the dose or concentration in dose–response relationships is typically shown on a semilog scale and dose–response relationships often show an S-shaped curve similar to chemical X in Fig. 1.2A. The data portrayed in Fig. 1.2A provide an example of a threshold dose–response relationship. In essence, while lower doses do not elicit toxicity, at some threshold level of exposure, a toxic response is noted (in this case in a proportion of individuals) which then increases in incidence with higher doses (or increases in magnitude when the degree or extent of a response is measured). The concept that a threshold exists in exposures below which no toxic response occurs has been the foundation for chemical risk assessments and regulatory decision-making for decades. It is assumed that if levels of exposure below the threshold do not elicit toxicity, then regulating/managing chemicals such that exposures fall below the threshold will maintain public safety and environmental health.

Figure 1.2 Basic types of dose–response relationships.

A threshold (A) and no threshold (B) dose–response relationship is shown. The threshold dose–response relationship has been the cornerstone for regulating noncarcinogens while the no threshold dose–response relationship is generally considered in estimating risk for genotoxic carcinogens.

Several conclusions can be extracted from threshold dose–response data. First, when comparing chemicals X and Y (Fig. 1.2A), one can see that chemical X is more potent, that is, it elicits toxicity at lower levels of exposure. If you draw a line at the 50% response level, you can graphically estimate the dose of chemical X that would elicit toxicity in 50% of the individuals (around 1 mg/kg/day). Similarly, the dose of chemical Y that elicits toxicity in 50% of the individuals can be estimated at about 10 mg/kg/day. Thus you can consider based on the toxic response being measured that chemical X is roughly 10 times more potent than Chemical Y. Second, both chemicals can elicit the toxic response in essentially all of the individuals exposed, as long as the dose is high enough. Third, these types of data allow you to operationally define a no effect or no observed adverse effect level (NOAEL). For a given dataset (in the case of Fig. 1.2A, doses of 0.01, 0.03, 0.1, 0.3, 1, and 3 mg/kg/day), the highest dose in the study associated with no toxicity is defined as the NOAEL. For chemical X, the NOAEL would thus be defined as 0.03 mg/kg/day, while the NOAEL for chemical Y would be 1 mg/kg/day. Chemical-specific NOAEL values derived primarily from experimental studies on chemicals that exhibit threshold dose–response relationships, along with considerations of uncertainty based on extrapolating results from animal studies to humans, and variability among different people, have historically been essential in estimating safe levels of exposures and protecting public health.

In contrast, Fig. 1.2B shows the second major type of dose–response relationship, that is, one in which no apparent threshold is exhibited. In this case, as before, increasing dose leads to an increased proportion of individuals exhibiting toxicity, but there is no clear-cut break between exposures that do or do not elicit toxicity. Genotoxic carcinogens often exhibit nonthreshold dose–response relationships. Even very low exposures may elicit some incidence of toxicity. The process for evaluating risk of chemicals that do not show a threshold is conducted by a different paradigm compared to those that show thresholds, based at least partly on the uncertainty of responses at very low levels of exposure, which are very difficult to study in experimental models for a variety of reasons.

Two substances with exceedingly different toxic potencies can be used to illustrate how both the chemical’s inherent properties and the type of exposure interact to influence whether or not toxicity occurs. Let us first consider botulinum toxins. These toxins exist as a family of eight distinct polypeptides (referred to as types A–H) that are produced by the bacterium, Clostridium botulinum and/or related microorganisms. Severe muscle paralysis is a potentially lethal response to botulinum toxin exposure. Nerve cells in complex organisms communicate with other neurons (and other cell types, e.g., muscle cells) by releasing specific neurotransmitters which interact directly with the target cell (see Chapter 6: Disruption of extracellular signaling and Chapter 20: Nervous system). All subtypes of botulinum toxin act by binding to specific proteins within the nerve terminal to block neurotransmitter release and thereby disrupt cellular communication.⁵ Neurons that supply or innervate skeletal muscles release the neurotransmitter acetylcholine to cause that muscle cell to contract. A botulinum toxin acting on those neurons will therefore block acetylcholine release, leading to reduced muscle contractions and potentially paralysis of the affected muscles.

Botulinum toxin A is considered the most toxic substance known to man, with reported LD50 values in the low ng/kg range (i.e., an amount approximately 100 trillion-fold lower than the weight of a human).⁶ It would therefore make inherent sense to avoid any exposure to these exceptionally toxic substances. As is well known however, botulinum toxins have been developed as therapeutic agents to reduce muscle contractions in disorders that are associated with excessive muscle contractions. Moreover, therapeutic applications for botulinum toxins to treat other medical conditions continue to be pursued.⁷ Thus the most potent toxic substances in the world can be used effectively and safely, but only by understanding their inherent toxic potential and by strictly controlling exposure.

On the other end of the spectrum from botulinum toxins is water, an absolutely essential substance for all living organisms on Earth. One would assume that any hazard associated with systemic water exposure would be minimal, and that is in fact, generally the case. Water is not without an inherent capacity to do harm, however. A reduction in blood sodium levels (hyponatremia) by excess water consumption can increase fluid uptake due to disruption of the sodium concentration gradient between blood and the organs/tissues. If excess fluid accumulates in the brain, swelling of the tissue will lead to increased pressure (due to the rigid, bony skull) and damaged/dead cells within the brain, potentially leading to severe effects including seizures, unconsciousness, respiratory arrest, and death.

Excessive water consumption has been reported in attempts to dilute a person’s urine before a drug test, leading to serious complications.⁸ Although infrequent, cases of child abuse have been reported involving forced water consumption and subsequent water intoxication.⁹ Some case studies report excessive water intake and water intoxication in marathon runners after a race. What is clear from these examples is that although water is absolutely essential for all living organisms, excessive intake (as with any substance) can lead to toxicity. Botulinum toxins and water therefore provide evidence that on the one hand all chemicals are toxic, and on the other even the most toxic substances can be used safely.

The extreme case of water intoxication provides the opportunity to consider a third type of dose–response relationship, one that is exhibited by substances which are essential for the organism. Fig. 1.3 shows a hypothetical dose–response relationship for water intoxication. Very low water is associated with dehydration, with fluid levels insufficient to maintain homeostasis, tissue hydration, ionic balances, and sufficient blood volume, leading to some form(s) of toxicity. Within a certain range of higher exposures, fluid homeostasis is maintained and no adverse effects are noted. With excessive (much higher) exposures however, adverse effects occur which can be life-threatening.

Figure 1.3 A U-shaped dose–response relationship.

This type of relationship is exhibited by essential substances.

Other types of dose–response relationships can be observed. For example, some endocrine disrupting chemicals (see Chapter 17: Organ system effects: endocrine toxicology) have been reported to elicit toxicity at low levels of exposure, but not at higher levels. Some chemicals can elicit beneficial effects at low levels of exposure, but adverse effects with higher exposures. These other nonmonotonic dose–response relationships may be based on adaptive changes (e.g., receptor upregulation or downregulation) or feedback loops that occur at one end of the dosing spectrum, but not at the other.

Botulinum toxin(s) as an acute toxicant is in a class of its own based on acute lethality (LD50 approximately 1 ng/kg). Ethyl alcohol (ethanol) is a common substance that is well known for its adverse effects with acute and long-term exposures. The adverse health effects of chronic alcohol consumption take a huge toll on many individuals, families, and society in general. Although it is possible to elicit severe toxicity with high acute ethanol exposures (and reports of hazing-related deaths from alcohol-related toxicity continue as evidence), its potency as an acute toxicant is actually very low (LD50 on the order of 5–10 g/kg, or over a billion times less potent than botulinum toxin). Table 1.1 shows a categorical ranking of acute lethality that can be used as a framework for comparing relative acute potencies. Note that botulinum toxin would be an outlier in this table, needing its own category (e.g., super-super toxic).

Table 1.1

Testing for toxicity is an essential component in the process of developing new drugs. Any new drug candidate must have its potential toxicity fully characterized before it is approved and introduced into the market. In preclinical or nonclinical drug testing, a number of methods are used to measure the desirable drug effects (i.e., its efficacy) as well as identify types of toxic responses that may occur with its use. The LD50, MTD, or NOAEL for a drug candidate can all be quantitative indicators for its relative potential to cause harm. When compared to a quantitative measure of its efficacy, the drug’s potency at causing desirable versus undesirable effects can be estimated as some form of a therapeutic index (TI). A common way to calculate TI is to divide the chemical’s LD50 by the dose of the drug that elicits a therapeutic response in 50% of the population, that is, the effective dose 50 (ED50): TI=LD50/ED50.

If we consider hypothetical dose-related data generated in a drug testing laboratory, we can further clarify the concepts of potency and efficacy. Dose-related studies to evaluate relative toxicity and efficacy can provide drug candidate-specific information important in selecting candidates for further development. One can see that high TI values for a given drug candidate would be advantageous over other candidates with lower TI values.

A relatively high acute LD50 or other indicators of acute toxicity means that the chemical in question has relatively low potency at eliciting acute toxicity. It must be stressed however that low toxicity with acute exposures does not mean that the chemical would be relatively safe with long-term exposures. Vinyl chloride, one of the highest production volume chemicals in the world, is extensively used in the production of polyvinylchloride-based plastics. In animal studies, vinyl chloride is slightly toxic based on the general acute toxicity scale shown in Table 1.1 (i.e., it has an LD50 in rats with oral dosing of about 4 g/kg). With long-term exposures, however vinyl chloride can elicit liver cancer in animals and humans.¹⁰ In a similar situation, toxic responses in ecological settings are rarely acute and tend to be characterized as reductions in growth, reproduction, and development. Consequently, such sublethal responses with environmental contamination tend to have more ecological relevance with regard to population changes than with the fate of individual organisms.

Another example of a chemical with low acute toxicity but which elicits chronic toxicity is the organophosphorus chemical, tri-ortho-cresyl phosphate (TOCP). TOCP is one isomer of a mixture of tri-cresyl phosphate (TCP), used for decades as a lubricant and plasticizer. The acute LD50 for TOCP is >1 g/kg. This slightly toxic chemical based on acute lethality can lead however to irreversible damage in the nervous system. Interestingly, the other two isomers (meta and para) in the TCP mixture also have low acute toxicity potential, but they cannot elicit the long-term neurological changes associated with exposure to the ortho isomer. Moreover, in contrast to vinyl chloride where repeated, long-term exposures are necessary to elicit chronic toxicity (liver cancer), the chronic effects of TOCP can occur following a single exposure. Fortunately, the ortho isomer is now removed from TCP in use today. It should be noted that some studies suggest that TCP (free of the ortho isomer) may contribute to another condition referred as aerotoxic syndrome. As a lubricant component in jet engines, TCP can leach into the aircraft cabin when an engine seal is defective, thereby exposing travelers and flight personnel to TCP vapors. A causal relationship between TCP and any aerotoxic syndrome has not been firmly established however.

1.2.2 Time as a factor in the expression of toxicity

The amount of time between exposure to a chemical and a toxic response is important in characterizing chemical toxicity. In acute poisoning, the interaction between time of exposure and dose on toxic outcome was studied by Fritz Haber, who was awarded the Nobel Prize for inventing the method to synthesize ammonia from nitrogen in ambient air. Haber was also the scientific adviser of the German Kaiser during World War I (WWI). Haber showed that the cumulative lethal effect of a toxic gas depended on the atmospheric concentration multiplied by the duration of exposure. These results were unfortunately used in the proposal to use chlorine gas as a chemical weapon in WWI. The study of the relationship between concentration and exposure time with acute toxicity of gases continues today.

Basic categories of toxic responses relative to the time of exposure include acute versus chronic toxicity, and immediate versus delayed toxicity. Acute toxicity is generally characterized by a rapid course of overt signs, generally occurring soon after the time of exposure. The harm from this type of toxic response is generally reversible, if the exposure is low enough for survival. Acute toxicity is also generally much easier to associate with a specific toxicant due to the relatively short amount of time for other factors to confound the interpretation of cause-and-effect.

An example of an acute intoxication would be the expression of neurological, muscular, and respiratory effects that occur shortly after acute exposure to an organophosphorus nerve agent such as sarin. Unfortunately, there are recent real-world examples of the type of acute toxicity that can be elicited by organophosphorus nerve agents.¹¹–¹³ The signs and symptoms of nerve agent intoxication, along with verification of chemical residues in environmental media or biomarkers of exposure in affected individuals in these cases, helped confirm a cause–effect relationship.

In contrast, chronic toxicity, either from an acute intoxication or from repeated lower level exposures, is often associated with an accumulation of damage over time. Chronic toxicity is generally more insidious in nature than acute toxicity, being more difficult to associate with a particular substance, and often characterized by irreversible damage. For example, the association between long-term exposure to tobacco smoke and chronic health consequences was only firmly established after decades of research (and unfortunately, facilitated by the large number of individuals affected).

1.2.3 Time as a factor in exposures

As stressed earlier, hazard is a product of both the intrinsic properties of a substance and the degree or extent of exposure(s). As with the role of time in the expression of toxicity, time is also important in characterizing exposures. In mammalian toxicity testing, acute exposures are either single or a few multiple exposures, all occurring within a short time period (up to 24 hours). Acute oral toxicity is most often based on studies with only single exposures. In some cases, for example, studies related to pesticide exposures in the diet, acute exposures can be throughout a given day (such as to model three meals). Subacute exposures are repeated exposures that occur roughly within a month. In mammalian toxicity testing, subacute exposures are often daily exposures occurring for 14 continuous days. Subchronic exposures are generally repeated, daily exposures occurring for 1–3 months. A subchronic dosing study in rodents typically lasts for 90 days. Finally, chronic exposures are repeated exposures that occur for more than 3 months, typically 6–24 months. Keep in mind that a chemical may elicit very different responses when lower exposures occur over longer periods, compared to responses following higher, short-term exposures. In the ecological testing context, acute exposures are generally 48–96 hours in duration, and chronic exposures cover an entire life or reproductive cycle. For example, chronic invertebrate bioassays can be as short as 10 days, but tests in fish can last up to 28 days, depending on the species.

1.2.4 Local versus systemic toxicity

The site of a toxic response is also an important characteristic in defining toxic potential. Local toxicity, that is a toxic response that occurs at the site of chemical contact, is very important in occupational settings. The majority of intoxications in the workplace involve dermal reactions, occurring at the site of chemical exposure on the skin. For example, acid spills can lead to caustic damage to the affected area of the skin, with relatively few systemic effects. Similarly, strong bases such as cationic detergents can damage the skin, buccal cavity, esophagus, or other areas of the gastrointestinal tract with relatively little systemic toxicity. Locally acting toxicants harm the tissues that are in direct contact (see Chapter 23: Toxicology in the home, and Chapter 24: Toxicology in the workplace).

While localized responses can be life-threatening, most severe intoxications involve absorption and systemic toxicity. Chemicals which are absorbed into the circulation can have far-reaching effects in tissues distant from the site of chemical contact. Organisms within aquatic ecosystems can undergo local toxicity at the site of absorption (i.e., gills), but generally systemic exposure is largely dependent on the solubility of the chemical in the water. Chemicals which are poorly soluble in water can still undergo uptake through dietary exposures, potentially leading to bioaccumulation.

Depending on the physical nature of the chemical, it may gain access into the circulation by which it can be distributed throughout the body. Once absorbed, a chemical can be modified by biotransformation reactions that alter the structure of the toxicant. Either the parent compound or a metabolite may interact with target macromolecules within the body to initiate a toxic response. Ultimately, the parent compound and/or its metabolites are eliminated by excretory pathways. If a chemical is poorly metabolized and accumulates within an organism, the likelihood of adverse effects is enhanced, as is the ecological transfer to predatory organisms that feed upon the contaminated organism. This process is referred to as biomagnification and occurs for several well-known persistent environmental contaminants including DDT (dichlorodiphenyltrichloroethane) and methyl mercury.

1.2.5 Interactions

The great majority of information known about chemical toxicity has been derived from experimental studies of individual chemicals. In contrast, individuals and communities/populations of individuals are typically exposed to a mixture of chemicals at any given time. These chemicals can come from the ambient environment or from dietary, occupational, or pharmaceutical sources. Understanding the toxicity of any chemical is complex and requires extensive investigation: the study of mixtures of chemicals can be markedly more complex. While a considerable amount of information on the mechanism of toxicity, toxicokinetics, biotransformation, etc., is known for a relatively large number of chemicals, much less is known about the toxicity of mixtures of chemicals.

A starting point for studying mixtures is with the interaction between two chemicals.¹⁴ Fig. 1.4 shows four basic types of interactions that can occur between two chemicals: additivity, antagonism, synergism, and potentiation. To illustrate these types of interactions, let us consider two chemicals referred to as A and B. For measuring toxicity, we will use a ranked grading scale for recording the severity of response from 0 (i.e., no signs of toxicity) to 6 (lethality). If chemical A alone elicits an average response of 2 and chemical B also elicits a score of 2, and when given together they yield a median score of 4, then one could conclude additivity. A simple description of additivity is:

Figure 1.4 Basic types of toxicological interactions between two chemicals.

Interactions leading to additivity, antagonism, synergism, and potentiation are illustrated.

With antagonism, the toxicity of one chemical is reduced or eliminated by the other. To use our earlier example of toxicity ranking, if chemical A elicits a median response of 5 while chemical B elicits no response (i.e., median score 0), but both chemicals given together yield a median score of 1, then one can conclude that chemical B was an antagonist of chemical A. A simple mathematical description for antagonism is:

Synergism occurs when two chemicals elicit some degree of a toxic response but when given together, the response is much greater than expected based on the toxicity of the individual compounds. For example, if both chemical A and chemical B elicit a median response of 1 but when given together they elicit a median score of 6, synergism has occurred. Potentiation is similar to synergism in that the toxic response to a chemical is amplified by another, but in this case, the amplifying chemical does not elicit the toxic response when given alone. Based on our scale, this would be exemplified by a median score of 3 for chemical A, a median score of 0 for chemical B, but a score of 6 when the two chemicals are given in combination. The simple algebraic description for both synergism and potentiation is:

A real-world example of potentiation was observed with the common organophosphorus pesticide malathion. While the use of organophosphorus pesticides has been reduced in the United States over the last couple of decades, malathion is still a very common insecticide in the United States and throughout the rest of the world. In 1976 a mosquito control operation in Pakistan using malathion in backpack sprayers led to unexpected human intoxications.¹⁵ Signs of toxicity were noted in applicators using one of three different formulations and were correlated with erythrocyte cholinesterase reduction in affected workers. It was later determined that all the formulations associated with toxicity contained a minor impurity, isomalathion. While isomalathion was relatively nontoxic, it blocked an enzyme (a carboxylesterase) that inactivated malathion. Blocking the breakdown of malathion by the impurity potentiated the toxicity of malathion.

1.2.6 Adaptations

As alluded to above, chemicals may elicit different types of toxic responses following acute versus repeated exposures. Living cells/organisms can respond dynamically to chemical exposures such that toxicity changes as the living system experiences and responds to the chemical over time. Tolerance refers to an adaptive decrease in response to a toxicant following previous exposures either to the same or a related chemical. There are two basic mechanisms for tolerance, dispositional and cellular.

Dispositional tolerance is exhibited as a decrease in toxic response based on a reduction in the level of the toxicant at the initiating site (receptor). Through changes in uptake/transport, biotransformation, distribution, or elimination, less of the chemical is available to interact with the receptor to initiate toxicity following prior exposure(s). A classic example of dispositional tolerance is shown by a reduction in sleep time induced by a sedative hypnotic such as phenobarbital. With repeated exposures, metabolic enzymes are induced that then more effectively inactivate phenobarbital, such that with additional exposures the chemical is more effectively eliminated and sedative effects are reduced.

In contrast, in cellular tolerance there is no change in the levels of the toxicant itself, but the cells/tissues where the toxicant acts in some way adapt such that the magnitude of the response is reduced. In many cases, this adaptation is due to changes at the receptor level. A general phenomenon exhibited by many types of receptors is dynamic changes in receptor density by prolonged exposure to either a receptor activator or a receptor blocker. While some toxicants can directly activate or block a receptor, receptor regulation can also be elicited by toxicant-induced changes in the levels of the endogenous signal for that receptor (see Chapter 6: Disruption of extracellular signaling). Regardless of whether it is a direct or indirect action, cells generally reduce their receptors following persistent receptor activation, and conversely increase their receptors with prolonged receptor blockade. Through these dynamic regulatory processes, homeostasis of the signaling pathway can be maintained.

References

1. Wax PM. Historical principles and perspectives. In: Hoffman RS, ed. Goldfrank’s toxicological emergencies. New York: McGraw-Hill Education; 2015;1–15.

2. Robb GL. The ordeal poisons of Madagascar and Africa. Bot Mus Leafl (Harv Univ). 1957;17(10):265–316.

3. Webster C. Paracelsus: medicine, magic, and mission at the end of time Yale University Press 2008.

4. Degrandjean P. Paracelsus revisited: the dose concept in a complex world. Basic Clin Pharmacol Toxicol. 2016;119:126–132.

5. Ferrari A, Manca M, Tugnoli V, Alberto L. Pharmacological differences and clinical implications of various botulinum toxin preparations: a critical appraisal. Funct Neurol. 2018;33(1):7–18.

6. Schantz EJ, Johnson EA. Properties and use of botulinum toxin and other microbial neurotoxins in medicine. Microbiol Rev. 1992;56(1):80–99.

7. Fonfria E, Maignel J, Lezmi S, et al. The expanding therapeutic utility of botulinum neurotoxins. Toxins. 2018;10:208 https://doi.org/10.3390/toxins10050208.

8. Klonoff DC, Jurow AH. Acute water intoxication as a complication of urine drug testing in the workplace. J Amer Med Assoc. 1991;265(1):84–85.

9. Metheny NA, Meert KL. Water intoxication and child abuse. J Emerg Nurs. 2018;44(1):13–18.

10. Brandt-Rauf PW, Li Y, Long C, Monaco R, Kovvali G, Marion MJ. Plastics and carcinogenesis: the example of vinyl chloride. J Carcinog. 2012;11:5.

11. Holstege CP, Kirk M, Sidell FR. Chemical warfare Nerve agent poisoning. Crit Care Clin. 1997;13:923–942.

12. John H, van der Schans MJ, Koller M, et al. Fatal sarin poisoning in Syria 2013: forensic verification within an international laboratory network. Forensic Toxicol. 2018;36:61–71.

13. Paddock RC, Sang-Hun C. Kim Jong-nam was killed by VX nerve agent, Malaysians Say. nytimes.com; 2017, February 23.

14. Pope C. Chemical interactions. In: 3rd ed. Elsevier Inc., Academic Press 2014;793–794. Wexler P, ed. Encyclopedia of toxicology. vol. 1.

15. Baker Jr EL, Warren M, Zack M, et al. Epidemic malathion poisoning in Pakistan malaria workers. Lancet. 1978;1(8054):31–34.

Chapter 2

Absorption, distribution, and excretion in complex organisms

Lara Maxwell,    Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK, United States

Abstract

If the dose makes the poison, then the concentration of a toxicant at its site of action will dictate the magnitude and duration of adverse effects. For a xenobiotic to reach the systemic circulation, it must move through or between the epithelial cells at the site of entry. Both passive diffusion and active transport facilitate absorption. The physicochemical properties of the xenobiotic govern its distribution, in combination with the anatomical and physiological characteristics of the organism. As sufficient concentrations of the xenobiotic reach the target site, adverse effects ensue. Numerous body systems exist to detoxify and eliminate the offending toxicant, such as renal, biliary, and respiratory excretion and metabolism. As the toxicant is cleared from the body, xenobiotic concentrations in tissues fall, terminating reversible toxic effects. Taken together, absorption, distribution, metabolism, and excretion encompass the ADME processes and contribute to the extent and length of toxicity.

Keywords

ADME; disposition; toxicokinetics; xenobiotic; rate constants; transporters

2.1 Introduction to xenobiotic disposition

Toxicants are xenobiotic molecules that produce adverse effects at their site of action in the organism’s body. The concentration of a toxicant over time at its site of action determines the magnitude of its effect, thus the famous quote by Paracelsus (CE 1492–1541) that …all things are poison and nothing (is) without poison. Solely the dose determines that a thing is not a poison.¹ For the toxicant to reach its site of action and produce its toxic effect, the xenobiotic must make contact with the susceptible parts of the body, such as the liver or brain. Once the molecule has reached the site of toxicity, many toxicants must enter the cells to interact with the nucleus, mitochondria, or other site of toxic action. Even substances that produce effects at the most superficial cutaneous structures must penetrate the outer layers of cells. For the xenobiotic to reach its site of action deeper inside the body, it must pass through multiple barriers to absorption and then be moved by the circulatory system to other tissues. Xenobiotics selectively make their way into, around, and ultimately out of the body via absorption, distribution, metabolism, and excretion, or the ADME processes.

2.1.1 Barriers and facilitators of xenobiotic movement

Cells are encased in phospholipid bilayer membranes, consisting of two phospholipid layers with their lipophilic tails sandwiched between inner and outer hydrophilic, phosphate heads. The lipid bilayer is a semipermeable membrane that excels at repelling polar or charged molecules and represents the primary barrier to xenobiotic movement into and out of cells.

2.1.2 Uptake, distribution, and elimination in complex organisms

For systemic toxicity to occur in complex, multicellular organisms, a xenobiotic must be absorbed into the circulation. In lower species, this may be simple interstitial fluid, hemolymph, or another type of circulating fluid, whereas in higher species, the circulatory fluid is blood. In these higher organisms, xenobiotics reach the tissues of the body by moving passively within the circulatory system, traveling through arteries, veins, and capillaries. Xenobiotic movement between capillaries and interstitial fluid occurs readily because most capillaries have loose attachments between the endothelial cells lining blood vessels, allowing xenobiotics to freely move between adjacent endothelial cells and into interstitial fluid. The concentration gradient, or difference in xenobiotic concentration between two different compartments separated by a semipermeable membrane, is the driving force responsible for the passive movement of xenobiotics into, around, and out of the body. This concentration gradient therefore governs the rate of flux, or that rate of movement across a semipermeable membrane. However, anatomical characteristics of the organism, as well as the physicochemical properties of the xenobiotic itself, also govern the rate of flux. Adolf Fick was a German physicist and physician who, among other interests, sought to describe the driving force of diffusion. Fick’s equation can be reworked to predict xenobiotic flux as dependent upon the concentration gradient and several anatomical factors²:

D is the diffusion coefficient and P is the lipid:water partition coefficient of the xenobiotic, whereas SA is the surface area, Cout − Cin is the concentration gradient, and ΔX is the thickness of the semipermeable membrane. Small molecular size of the xenobiotic increases D, and lipophilicity increases P. Consequently, small, lipophilic xenobiotics tend to have higher flux. The plasma:tissue partitioning coefficients can be determined from physicochemical drug properties, such as by its octanol:water partition coefficient along with other characteristics, or determined experimentally by measuring the concentrations in each tissue of interest. Such partitioning coefficients describe which tissues will harbor the intoxicant at the highest concentrations, as well as being essential to building physiologically based pharmacokinetic models.

Within the body, tissues that have a large SA, such as the alveoli of the lung or the villi and microvilli of the small intestine, have higher flux. Thin tissues also have higher flux as compared to that of thicker tissues. Again, the alveoli of the lung are an area of rapid xenobiotic flux due to both its large surface area and thin alveolar and capillary membranes, properties that allow rapid gas exchange.

2.1.3 Ion trapping

Since the physicochemical properties of the xenobiotic affect its flux, xenobiotics that can be ionized at physiological pH can differ in lipophilicity at different anatomical sites with differing pHs. In the case of these weak acids and weak bases, the ionized form is unable to readily diffuse across the lipid barrier of cell membranes, whereas the unionized form can reach equivalent concentrations between compartments separated by a semipermeable membrane. Therefore, the equilibrium between the ionized and unionized forms determines whether the xenobiotic can substantially cross these membrane barriers. The Henderson–Hasselbalch equation:

where pKa is the equilibrium dissociation constant for an acid and is the pH at which half of the molecules are ionized and half are unionized. This equation can be used to calculate the proportion of the xenobiotic that is present in its ionized and unionized states. This ratio of ionized:unionized (weak acid) or unionized:ionized (weak base) is used to determine whether a particular tissue will favor the ionized or unionized form as compared to plasma, with its nearly neutral pH of 7.4. For example, the proportion of a weak base that will be ionized in milk, which is acidic relative to plasma, can be predicted and used to calculate the relative amounts present in plasma and milk at equilibrium (Fig. 2.1). From this calculation, we can demonstrate that the weak base will be trapped in the more acidic environment of the milk. Conversely, a weak acid will be trapped in a more basic environment, such as occurs when pentobarbital is trapped in alkalinized renal ultrafiltrate (Fig. 2.2). As a weak acid, pentobarbital exists in either ionized state, which has a negative charge and so is unable to pass through the cell membranes lining the renal tubule, or its unionized state, which is able to pass through cell membranes and be resorbed from the tubule and into blood. Alkalinizing the urine, therefore, encourages the ionized form of pentobarbital to predominate, and so it passes out into urine faster than if the urine pH was not modified. If more pentobarbital is excreted in urine, then there is less present in the body, so signs of toxicity will wane faster. Whether it is a weak acid or a weak base, any ion that is trapped by the pH of an environment is said to be subject to ion trapping.

Figure 2.1 Proportion and amounts of a weak base distributing into milk at equilibrium.

Figure 2.2 Ion trapping will enhance the elimination and excretion of pentobarbital into urine when the urine is alkalinized by the administration of bicarbonate.

2.1.4 First-order rate constants

Where Fick’s equation (mentioned earlier) adequately explains xenobiotic flux, xenobiotics move with their concentration gradients, and the rate of transfer between compartments can be mathematically described by first-order rate processes (Fig. 2.3). Here, rate constants, such as K01, describe the constant proportion of the xenobiotic per unit time that moves across a semipermeable membrane, or aggregate of membranes, that separates compartments (Fig. 2.4).

Figure 2.3 Flux versus concentration for first-order (solid line) and capacity-limited or zero-order (dashed line) rate processes.

Figure 2.4 First-order rate transfer from gut lumen to blood after oral administration of a xenobiotic.

This first-order rate constant is inversely related to a half-life of transfer, since:

The resulting half-life is then the time it takes for one-half of the xenobiotic to be transferred from one compartment to the other. More specifically, the term elimination half-life is often used to describe the time it takes for one-half of the xenobiotic to be eliminated from the plasma (Fig. 2.5).

Figure 2.5 Elimination half-life describes the time it takes for the plasma xenobiotic concentration to decline by one-half.

2.1.5 Xenobiotic transporters

In addition to the simple diffusion that allows a xenobiotic to move with its concentration gradient from areas of higher to those of lower concentration, some xenobiotics may be substrates of specific transporters. As their name implies, the ATP-binding cassette, or ABC transporters, use energy to actively transport their substrates. The ABCB1 subfamily of transporters serves a generally protective role, preventing xenobiotics from being orally absorbed at the apical surface of the enterocyte, from entering sensitive tissues such as at the endothelial cell of the blood–brain barrier, or facilitating removal of the xenobiotic into urine or bile at the proximal tubule cell or hepatocyte, respectively. Of course, these transporters can pose problems in drug therapy, such as the discovery of ABCB1 transporters that were once referred to as multiple-drug resistance proteins (MDR), because they actively pumped chemotherapy drugs out of cancer cells, conferring drug resistance to such cells.³ Similarly, multidrug resistance-associated proteins and breast-cancer resistance proteins (ABCG2, BCRP) can confer resistance to anticancer drugs.⁴ Inhibitors of such pumps, for example, elacridar, allow these drugs to reach higher intracellular concentrations and may restore drug efficacy against cancer cells.⁴ Diversified genes for ABC protein superfamilies have also been found in aquatic invertebrates, with possible adaptation of ABC genes due to environmental pressure from aquatic pollutants.⁵ These transporters, particularly multixenobiotic resistance transporters, have protective effects in a variety of invertebrates, but can also be themselves inhibited by environmental pollutants.

Another important class of transporters, the solute carriers (SLC), are passive transporters, symporters, and antiporters.⁶ Members of this group include SLC22, which contains the well-known organic anion (OAT) and organic cation (OCT) transporters. As the name implies, OATs have a predilection for the transport of anions, whereas OCTs are more likely to transport cations, though there is a surprising degree of overlap between the specificity of OATs and OCTs. These transporters are also generally protective, and their location at the renal epithelial cells lining the proximal tubule of the kidney promotes the removal of ionized xenobiotics from blood and into the renal ultrafiltrate. Another important SLC group includes SLC47, the MATEs. The organic anion transporting polypeptides (OATP transporters) are numerous and less well-defined, but transport many xenobiotics into and out of the cells of excretory organs, such as the renal epithelial cells and hepatocytes. Divalent metal transporters (DMT1, SLC11A2) are located in enterocytes, where promiscuous transport of divalent metals facilitates the uptake of several toxicants, including mercury and lead. Amino acid transporters (LAT1, LAT2, SLC7) may also facilitate heavy metal absorption and movement across the blood–brain barrier.

2.1.6 Saturable kinetics

As opposed to the passive diffusion of xenobiotics described earlier that rely on a concentration gradient for xenobiotic flux, xenobiotic transporters and biotransformation depend on proteins. When there are relatively few xenobiotic molecules compared with the number of available transporters or metabolizing enzymes, the first-order rate constants will continue to adequately describe the rate of xenobiotic disposition. The key concept is that processes that make use of enzymes or transporters are saturable due to the limited, finite number of transporters or enzymes available. Once the majority of transporters or enzymes are occupied by the xenobiotic, the rate of transport or metabolism is capacity limited (Fig. 2.4), as described for metabolism by the Michaelis–Menton equation:

Here −dC/dt is the change in substrate concentration with respect to time, Vmax is the maximum velocity of the reaction, and Km is the Michaelis–Menton constant, which is the xenobiotic concentration at ½ of Vmax. At high xenobiotic concentrations, Vmax is the zero-order rate constant, where a fixed amount of xenobiotic is metabolized per unit time. At xenobiotic concentrations well below Km, a first-order rate constant (Vmax/Km) will apply. Similar saturable kinetics apply to any dispositional process that uses a transporter, carrier, or binding protein with a limited capacity that can therefore be saturated (Fig. 2.3).

2.2 Absorption of xenobiotics

The disposition of a xenobiotic in an organism can be described in terms of its absorption, distribution, metabolism, and excretion, collectively called the ADME processes. Absorption is the movement of the xenobiotic across the body’s protective barriers, ultimately entering the blood via regional capillaries. The xenobiotic can enter by various routes of exposure, with broad categories of parenteral, enteral, and other. Parenteral routes of administration usually connote deliberate exposure and include intravenous, intramuscular, subcutaneous, intraosseous, intraarticular, and epidural. Enteral routes of administration are most often oral, or per os, but feeding tubes and rectal administration would also be included here.

2.2.1 Enteral exposure

For oral routes of administration, ion trapping of weak acid or weak base xenobiotics can determine the region of the gastrointestinal tract where absorption occurs. For example, since the stomach lumen is a highly acidic environment with many protons, the Henderson–Hasselbach equation predicts that a weak acid will predominantly exist in its unionized state, and thus be able to diffuse across gastric cell membranes into interstitial fluid and blood, where the weak acid will be trapped in the more basic environment of the blood. Conversely, weak bases will be primarily ionized in the stomach, so will be trapped there and poorly absorbed. Instead, the more basic environment of the small intestine favors the weak base existing predominantly in its unionized state, thereby facilitating intestinal absorption.

Absorption by simple diffusion is governed by Fick’s equation. However, if enterocyte transporters are involved, then the rate of enteral absorption is determined by Michaelis–Menton kinetics. For example, if lead is administered into the intestinal lumen of chicks, then the percentage of the lead dose that leaves the lumen decreases with increasing lead concentration.⁷ Since the percentage of lead leaving the lumen and entering tissues is inversely related to dose, lead flux is saturable and fails to follow first-order absorption kinetics, where a fixed