Toxic Endpoints in the Study of Human Exposure to Environmental Chemicals

CHAPTER FOUR

Toxic Endpoints in the Study of Human Exposure to Environmental Chemicals Andrew D. Wallace Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina, USA

Contents 1. Introduction to Toxic Endpoints 2. Toxicity Testing 2.1 In vivo testing 2.2 In vitro testing 3. Carcinogenesis 4. Hepatotoxicity 5. Renal Toxicity 6. Neurotoxicity 6.1 Neurotoxic endpoints 6.2 Environmental neurotoxicants 7. Reproductive Toxicity 8. Endocrine Toxicity 9. Immunotoxicity: Respiratory 9.1 The immune system and immunotoxic endpoints 9.2 Immunotoxicants 10. Concluding Remarks References

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Abstract Human exposure to chemicals in the environment can occur in an acute or chronic manner causing toxicity to different organs or resulting in other adverse health effects. To assess if chemicals encountered by humans in different environments have the potential to be toxic, both in vitro and in vivo testing models can be utilized and will be discussed in this chapter. The structures and function of different organs of the body often predispose these organs to being especially sensitive to chemical exposures. Specificity, a general description of endpoints of toxic action will be discussed in relation to carcinogenesis, hepatotoxicity, renal toxicity, neurotoxicity, reproductive toxicity, endocrine toxicity, and immunotoxicity. Examples of environmental chemicals causing

Progress in Molecular Biology and Translational Science, Volume 112 ISSN 1877-1173 http://dx.doi.org/10.1016/B978-0-12-415813-9.00004-0

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toxicity will be provided, and endpoints will be discussed ranging from histopathological characteristics to gene expression profiling.

1. INTRODUCTION TO TOXIC ENDPOINTS Human exposure to chemicals in the environment can be acute or chronic, which can lead to toxicity. A chemical may alter biological pathways leading to human health consequences. Toxicity assessments aim to determine the potential of a chemical to have deleterious effects, the conditions under which this occurs, and the characterization of the chemical’s action. To determine if a chemical causes toxicity, many types of testing systems exist, and many different endpoints of toxic action may be considered. Testing can be divided into in vivo testing, which can include different animal models, or alternatively in vitro models can be utilized. Both approaches have inherent benefits and limitations. This chapter will give a general description of endpoints of toxic action using examples from environmental chemicals with relation to carcinogenesis, hepatotoxicity, renal toxicity, neurotoxicity, reproductive toxicity, endocrine toxicity, and immunotoxicity. Human health and risk assessment associated with toxic endpoints is covered in detail in Chapter 9.

2. TOXICITY TESTING 2.1. In vivo testing In vivo testing uses animal models such as rodents, rats or mice, or nonrodent models including rabbits or dogs. The simplest acute toxicity testing uses the endpoint of lethality of a chemical, finding the median lethal dose or LD50. Subchronic testing involves the dosing of animals at multiple concentrations over time periods such as 28 days or 90 days. Chronic studies of 1 or 2 years are typically done to determine carcinogenicity. Endpoints are measured during testing to assess impacts on parameters such as food intake, body weight, blood chemistry, and behavior, while postmortem assessments are made of tissues and organs including histopathology. In vivo testing of a chemical can assess the impact of different routes of exposure including oral, dermal, inhalation, or injection.1 Additionally, in vivo testing also takes into account and allows for determination of the toxicokinetics of a chemical including the absorption, distribution, metabolism, and excretion.

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2.2. In vitro testing The use of in vitro testing systems is an attractive alternative to in vivo testing due to the exorbitant cost of in vivo testing, long durations of testing paradigms, and overwhelming number of environmental chemicals that need to be tested. In vitro systems can be simple, such as individual enzymes or cellular extracts, or more complex systems that utilize immortalized cell lines or primary cell cultures. A chemical may be incubated with individual enzymes or cellular extracts containing many enzymes to determine the impact of the chemical on the functions of enzymes. For example, individual cytochrome P450 (CYP) enzymes or liver microsomes, containing multiple enzymes, may be incubated with a chemical to determine if the functions of enzymes are inhibited or activated. Alternatively, immortalized cell lines or primary cells can be exposed to the chemical, such as a liver cell line or hepatocytes. Multiple endpoints can be assessed, and the goal of in vitro testing is to aid in the understanding of the in vivo effects.2 Useful cytotoxicity assays assess endpoints such as membrane integrity, apoptosis, cellular proliferation, and mitochondrial function. In vitro testing can also assess the potential carcinogenic nature of a chemical, such as the Ames test. The US Environmental Protection Agency’s ToxCast program and the Tox21 collaboration with the National Toxicology Program and National Institutes of Health Chemical Genomics Center have taken the approach of using a large number of in vitro high-throughput screening (HTS) assays. The initial testing of the ToxCast utilized 320 chemicals, consisting mostly of pesticides, and 467 different in vitro assays.3 These assays were done over a range of doses and multiple time points and included cell-free and cellbased assays. A few of the assessed endpoints were enzyme inhibition, CYP induction, cytotoxicity, genotoxicity, receptor transcription factor activity, and gene expression profiling. The Tox21 initiative is an effort by NIH to test, using cell and biochemical-based quantitative high-throughput screening (qHTS), approximately 10,000 environmental chemicals and approved drugs to identify those that can alter biological pathways and may result in toxicity.4 The resulting large data sets from ToxCast will be analyzed by bioinformatic approaches, compared to data collected using traditional in vivo approaches, and will be incorporated into the US EPA Toxicity Reference Database (ToxRefDB). The resulting use of numerous in vitro tests will aid in prioritizing and predicting the hazards associated with environmental chemicals.

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3. CARCINOGENESIS Carcinogenesis is the process of the change of normal cells into neoplastic cells and subsequently into a tumor. A chemical or its metabolite(s) can interact with DNA, RNA, or proteins causing changes in normal cellular functions such as altered cellular proliferation. An environmental chemical can act as a carcinogen by genotoxic or nongenotoxic mechanisms. A genotoxic chemical causes carcinogenesis by events initiated by damaging interactions with genetic material. Such DNA-damaging agents are typically found to be mutagenic. Nongenotoxic chemicals are not mutagenic but act in ways other than directly modifying DNA to cause abnormal cellular growth. It should be recognized that carcinogenesis is a complex process that occurs in multiple stages including initiation, promotion, and progression.5 Regardless of the mechanism, we are exposed to many different chemicals in our diverse work and living environments that are carcinogens. There are many endpoints that can be assessed when considering if an environmental chemical is a carcinogen. While chemicals that cause carcinogenesis are separated into their modes of action as genotoxic or nongenotoxic, most known human carcinogens are genotoxic.6 The ability of genotoxic chemicals to alter DNA can be assessed using bacteria, yeast, and mammalian in vitro models in a battery of tests that consider many different endpoints.7 Genotoxicity assays generally assess endpoints such as point mutations, frame shifts, mammalian cell chromosomal damage, altered DNA repair, and cell transformation.5 A simple such assay is the Ames test, developed by Bruce Ames, which determines if a chemical causes DNA damage that will allow a specific bacteria strain to grow under conditions limited by an essential amino acid. This type of reversion assay tests if a chemical causes mutagenesis of the bacteria strain’s DNA, which will allow the bacteria to grow on a selective media. Other tests determine if a chemical, or its metabolites, directly forms DNA adducts, causes replication errors, repairs errors, or causes DNA strand breakage.8 Genotoxicity tests are more thoroughly covered by Preston7 and Woolley.6 While it is clear that genotoxic environmental chemicals that are mutagenic are serious human health concerns, it is more difficult to assess nongenotoxic chemicals. Considerations of species-specific responses and dose relevance need to be considered when determining if chemicals that act as nongenotoxic carcinogens in other species also pose a health threat to humans. Chemicals may cause cancer by nongenotoxic mechanisms that

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do not involve direct damage to DNA but may involve epigenetic processes.9 Chemicals can cause epigenetic changes by altering genomic DNA methylation patterns or chromatin structure, which leads to altered genome stability, gene expression, or cell cycle control. The impact of altered gene expression or signal transduction pathways leads to failure of the normal controls of cellular proliferation, differentiation, or apoptosis. Epigenetic changes such as hypomethylation or hypermethylation of specific regulatory genes or in a genome-wide manner are often seen in tumors and are also often seen in premalignant and early stages of cancer.10 It is becoming increasingly clear that cancer involves both genetic and epigenetic processes, and both components are involved in cancer development.11,12 Traditionally, rodent models are used to test if a chemical is a carcinogen by exposing the animal over a 2-year period at varying doses including a maximum tolerated dose. Tissues and organs are examined by histopathological methods to identify abnormal masses or lesions.6 There are also many types of accelerated models, such as transgenic mice, that have a shorter exposure period causing earlier tumor formation.13 These can be very expensive undertakings, and researchers are attempting to develop alternative approaches that are less costly in terms of time and money. One approach is the development of models whose endpoints are the identification of signature changes in gene expression by toxicogenomic methods that are predictive of cancer development.14,15 This approach utilizes shorter exposure times and attempts to identify early precancerous changes in gene expression. The approach of transcriptomic studies, using measurement of mRNA abundance, has been shown to be an important method to identify new pathways impacted by chemical exposures that have not been previously identified.16 As our understanding about the modes of action that lead to carcinogenesis improves, early key events will be recognized and used to identify environmental chemicals that cause carcinogenesis.8 For example, the key event of receptor activity after environmental chemical exposure was assessed in relationship to liver cancer formation in rodents and found that the chemicals linked to liver lesions enhanced receptor activity to a greater degree.17 Humans are exposed to carcinogens in all the environments, and a major goal is to lessen exposures in these environments. The US National Toxicology Program twelfth report on carcinogen lists a few hundred known or reasonably anticipated carcinogens.18 One of the earliest identified carcinogens was found to be polyaromatic hydrocarbons (PAHs), specifically one such PAH that has been extensively studied is benzo(a)pyrene, and exposure

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occurs in many human environments from exhaust fumes, cigarette smoke, and charbroiled food. In occupational environments, humans are exposed to many carcinogens such as benzene, formaldehyde, vinyl chloride, metals (arsenic, beryllium, cadmium, and nickel), coal by-products, mustard gases, benzidine and benzidine type dyes, and radiation.19 For outdoor workers, exposure to UV radiation and for transportation workers, exhaust fumes are carcinogens of concern. In the domestic environment, exposure to carcinogens occurs from exposure to chemicals in food, second-hand tobacco smoke and radon. Aflatoxins are fungal-produced mycotoxins that are carcinogens and contaminants of food, which is a serious health concern in many parts of the world where the food supply is not closely monitored. Pesticides and diesel exhaust are carcinogens that are found in the agroecosystem and deployment environments.18,20,21

4. HEPATOTOXICITY Hepatotoxicity is the result of a chemical that causes adverse effects to the liver, which is often a target tissue of toxicity for environmental chemicals. The structural architecture and functions of the liver make it uniquely susceptible to toxic insult. The liver is highly exposed to chemicals because chemicals taken up orally are absorbed from the gastrointestinal tract into the blood, and the blood then flows directly to the liver. One of the functions of the liver is to remove xenobiotics, and high concentrations of xenobiotic-metabolizing enzymes are present in the liver. Both xenobiotics and bile acids move from the blood and into bile ducts for excretion into the intestine. Bile acids and xenobiotics that are excreted can be reabsorbed in the gut and reenter the blood stream in a process known as enterohepatic circulation, which can concentrate toxicants. The first sign of liver toxicity is often the development of abnormal accumulation of lipid in the liver, known as steatosis or fatty liver. The actions of toxic chemicals can alter the normal uptake of lipids and release of triglycerides in the form of lipoproteins. In animal models, this can be clearly seen in histopathology sections of the liver as vacuoles of fat in hepatocytes, the main cell type of the liver. Another sign of liver toxicity that can be clearly observed in histopathology sections is the disruption of the normal energydependent bile flow known as cholestasis, which leads to jaundice. Hepatotoxic chemicals that injure hepatocytes can trigger specific cells of the liver to deposit collagen, proteoglycans, and glycoproteins in a healing process known as fibrosis. Chronic fibrosis leads to cirrhosis, which is characterized

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by irreversible damage to the normal liver structure, functions, and blood flow. Fibrosis can be clearly observed by histopathology as normal liver structures are filled with deposits of proteins known as formation of an extracellular matrix. Chemicals that are toxic to the liver can also cause cell death by either necrosis or apoptosis. Blood tests are commonly done to detect liver injury, and when cells of the liver are damaged, they release proteins that are expressed in liver cells. The enzymes alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (AP) are specifically expressed in liver cells. The presence of ALT and ASP in the blood indicates that hepatocytes of the liver have been damaged, while the presence of AP indicates damage to cells of the bile duct.22 Cholestasis can also be assessed using blood tests that measure the serum levels of bilirubin and bile acids. The use of gene expression studies has most recently attempted to identify specific patterns of regulated genes associated with hepatotoxicity due to specific classes of chemicals.23,24 This can be done by isolating mRNA from toxicant-exposed animals, or isolated hepatocytes, to determine an expression profile and identify biomarkers of exposure. This type of approach may provide valuable information about the mechanism by which a toxicant is causing toxicity, but also, the gene expression changes can be observed much earlier than the subsequent resulting liver toxicity.16 With xenobiotic exposure, there often is an increase in expression of liver xenobiotic-metabolizing enzymes that are associated with increased liver weight and changes in liver enzymes.25,26 This type of induction may be seen as an adaptive response to a toxicant insult, but when this response is excessive, or produces toxic metabolites, hepatotoxicity can result. Assessment of hepatotoxicity due to dioxin-like compounds were examined by gene expression profiling and identified shared changes in gene expression, linked with phenotypic liver changes, that also seemed share a similar mechanism of action via the aryl hydrocarbon receptor.27 The use of isolated human hepatocytes from multiple individuals can also identify genes commonly regulated by toxicant exposure but also can identify individual differences in responses that may be involved in individual susceptibility to hepatotoxicity.28 Potentially, analysis of blood samples to identify signature changes in the gene expression of blood cells that are predictive markers of liver injury may allow an even earlier assessment that a chemical causes hepatotoxicity.29 A classic example of a hepatotoxic chemical is the industrial chemical carbon tetrachloride (CCl4), which initially causes liver steatosis. CCl4 is

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metabolized by enzymes in the liver, creating a reactive free radical that leads to disruption of lipids, proteins, and nucleotides. This results in cellular necrosis, fibrosis, and cirrhosis.30 Besides CCl4, other halogenated aliphatic hydrocarbons found in EPA Superfund sites have been shown to be hepatotoxic such as hydrochlorofluorocarbons (HCFCs), chloroform, trichloroethylene, and certain other haloalkanes.22 Pesticides, such as paraquat and endosulfan, can be toxic to the liver, causing steatosis, necrosis, cholestasis, and elevation of ALT and AST enzymes.31,32 Pesticides can also alter liver xenobiotic metabolism by the inhibition or induction of liver enzymes resulting in potentially toxic interactions. Examples include organophosphorus pesticides, such as chlorpyrifos, endosulfan, and pyrethroids.33–36 Other extensively studied hepatotoxic agents include ethanol and the drug acetaminophen. Ethanol exposure leads to interference with normal hepatic lipid metabolism resulting in steatosis, as well as activation of immune cells of the liver causing necrosis and oxidative stress. Chronic ethanol exposure leads progressively to fibrosis and cirrhosis. Acetaminophen overdosing is quite common and can result in total liver organ failure. Hepatotoxicity results from liver xenobiotic metabolism of acetaminophen to a reactive metabolite that interacts with liver lipids and proteins resulting in necrosis.37 The severity of acetaminophen hepatotoxicity is also much greater when coexposure to ethanol occurs, as ethanol increases the levels of enzymes responsible for production of acetaminophen’s reactive metabolite. A number of naturally occurring toxins also can cause hepatotoxicity, including phalloidin and a-amanitin which are toxins found in mushrooms.38,39 After ingestion, a-amanitin is taken up by the liver cells and acts to inhibit RNA polymerase activity. a-Amanitin undergoes excretion in the bile and enters the enterohepatic circulation resulting in further liver damage. Many plant species also produce natural hepatotoxins.22 Aflatoxin B1 is a mycotoxin produced by the fungus Aspergillus that is hepatotoxic, causing necrosis, later cirrhosis, and is considered a hepatocarcinogen.

5. RENAL TOXICITY Renal toxicity is the result of xenobiotic exposure that damages the kidney.40 Like the liver, the unique structure and functions of the kidney lead to its susceptibility to toxic insults. The main functional unit of the kidney is the nephron, which consists of a glomerulus, proximal tubules, and distal tubules. The high level of blood flow; the process for forming urine, which concentrates toxicants; and active transport processes further

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concentrate the levels of toxicants; all contribute to the susceptibility of the kidney to toxicants. The process of forming urine involves filtering blood at the glomerulus, and as the filtrate moves through the tubules, water, glucose, and essential nutrients are conserved by being reabsorbed into the blood. This leads to any toxicant in the filtrate being greatly concentrated. Contributing to the susceptibility of the kidney is that specific segments of the nephron contain high level of xenobiotic-metabolizing enzymes that are bioactive potential toxicants. Toxicants in the blood can also move into the proximal tubules by active transport mechanisms, further increasing the concentrations of toxicants. Renal toxicity results in failure of the kidney to function normally, and therefore, toxicity can be determined by observed changes in the contents of urine. Increased levels of glucose, water, and proteins in the urine are all indications that excretory functions have been altered. Additionally, changes can be observed in the blood, such as blood urea nitrogen or serum creatinine levels. A number of noninvasive biomarkers of renal injury are proteins found in urine after kidney damage, such as kidney injury molecule-1 (KIM-1), b2-microglobulin, and albumin.41,42 The presence of albumin or other high molecular weight proteins in the urine suggests damage to the glomerulus. Damage to the proximal tubule cells leads to the release of the kidney-specific enzyme g-glutamyl transpeptidase, which can be measured in the urine. KIM-1 is an especially promising biomarker as this transmembrane protein is only expressed by proximal tubule cells after they are damaged.43,44 A portion of the KIM-1 is released into the urine, which is easily measurable, and the levels of KIM-1 correlates with the degree of proximal tubule injury as seen by histopathology.41 Damage to distal tubules is much harder to study, but concentration of toxicants can lead to precipitation of poorly soluble compounds out of solution leading to obstruction of urine outflow. While a large number of drugs cause nephrotoxicity, a number of environmental chemicals also target the kidney.45 The proximal tubules are especially susceptible to injury due to the expression of xenobioticmetabolizing enzymes, which can cause bioactivation of xenobiotic chemicals, and also expression of transport proteins, which can cause accumulation of toxicants. Occupational exposure to the metals, such as mercury and cadmium, leads to nephrotoxicity, which primarily involves the proximal tubules. Cadmium can form a complex that accumulates in kidney cells, and due to the long half-life of cadmium in humans, low exposure levels over years can lead to accumulation to toxic concentrations. Mercury from industrial or dietary sources is complexed in the body with molecules like

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glutathione, which is taken up and accumulates in proximal tubule cells. Conjugated forms of mercury structurally resemble amino acids, and transporters mistakenly actively take up these conjugates.46,47 Exposure to mercury or chromium results in an increase the levels of urinary KIM-1, and the level of this biomarker correlates with the degree of proximal tubule injury seen by histopathology.41 Chloroform (CHCl3) is a widely used industrial chemical that caused nephrotoxicity. Chloroform is metabolized to the reactive intermediate, phosgene, by xenobiotic-metabolizing enzymes. The resulting phosgene is highly reactive and acts on cellular molecules to cause cell damage. Hexachlorobutadiene is also nephrotoxic, but this industrial solvent is conjugated in the liver and accumulates in proximal tubule kidney cells where it is further metabolized to reactive intermediate by the kidney-specific enzyme cysteine conjugate b-lyase. Ethylene glycol is found in antifreeze and brake fluids, and its metabolite oxalic acid binds to calcium, forming a product which precipitates in distal tubules and blocks urine flow. Similarly, melamine and cyanuric acid co-contamination of pet and baby food in China led to the formation of crystals in kidney tubules, which has been recapitulated in animals.48 Aristolochic acids from the Aristolochiaceae family of plants are found in Chinese herbal remedies and have been found to be nephrotoxic to humans and in animal models, causing apoptosis of proximal tubular cells and fibrosis.49,50 Gene expression profiling of renal toxic agents have been undertaken to identify gene-based biomarkers of kidney toxicity, which were linked with histopathological observations.16,51 Alternatively, noninvasive approaches are being developed and have identified a number of candidate biomarkers to detect kidney toxicity due to drugs or environmental chemicals, including urinary KIM-1, neutrophil gelatinase-associated lipocalin (NGAL), and others.52 The long-term goal is that novel kidney biomarkers will predict at an early stage that a drug or an environmental chemical is causing nephrotoxicity before severe organ damage occurs.53

6. NEUROTOXICITY 6.1. Neurotoxic endpoints There are a large number of neurotoxic endpoints, both acute and chronic, and many of them are caused by neurotoxicants associated with human environments. Neurotoxicity has been treated in some detail by Blake54,55 and Section 6 owes much to these treatments. The following section is a brief summary of the importance of the environmental neurotoxicants.

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“Neurotoxicity refers to the ability of an agent to adversely affect the structural or functional integrity of the nervous system.”55 Essentially, all human environments contain neurotoxicants, organophosphorus insecticides (OPs) in the agroecosystem being the most obvious, although these OPs are also found in other occupational as well as the domestic and military deployment environments. For convenience, effects of chemicals on the nervous system are often classified as structural or functional, but it should be kept in mind that any adverse effect must—in the final analysis—result from a functional impairment. The most common structural effects are demyelination, axonopathy and neuronopathy. Demyelination. Myelin aids signal transduction in the nervous system by acting in a manner analogous to an electrical insulator and preventing loss of ionic currents. Neurotoxicants that target the synthesis or integrity of peripheral nervous system (PNS) myelin may cause numbness and tingling, muscle weakness, poor coordination, and paralysis. This disorder in the PNS is called myelinopathy. In the brain, it is known as toxic leukoencephalopathy. Clinical manifestations of toxic leukoencephalopathy are varied, extending from headaches through cognitive dysfunction to paralysis and death. Neurotoxicants that produce primary demyelination include those that affect the integrity of the myelin sheath without, or prior to, damage to the myelinating cells and those that injure myelin-producing cells. The former group is represented by hexachlorophene and the organotins, agents that cause reversible edema between the layers of myelin. Different nerves vary in their susceptibility to different agents, the optic nerve being, for example, susceptible to hexachlorophene, while other cranial nerves are more susceptible to solvents such as xylene or trichloroethylene. In contrast, chronic exposure to cyanide and carbon monoxide is thought to directly injure myelin-producing Schwann cell bodies in the PNS and oligodendrocytes in the CNS. Inorganic lead, a contaminant in the urban, domestic, and occupational environments, also damages myelin-producing cells. Oligodendrocytes appear more sensitive to lead toxicity than astrocytes or neurons, and the developmental effects of lead exposure may be preferential inhibition of oligodendrocyte precursor cell differentiation. Axonopathy. Axonopathy involves selective degeneration of the axon while leaving the cell body intact. In many cases, the most distal portions of the longest- and largest-diameter axons are most vulnerable to this type of toxicity, and these areas degenerate first. With continued exposure to the toxicant, however, the degeneration progresses proximally and may

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eventually affect the entire neuron. This distal-to-proximal degeneration is called “dying back neuropathy.” As the axon degenerates, the myelin associated with it also breaks down, although in the PNS, Schwann cells may survive and guide regeneration of the axon in the PNS. Axonal regeneration does not, however, occur within the CNS. The reason for the enhanced vulnerability of distal axons to toxic effects may be due to the fact that these regions are more dependent on intact energy-dependent axonal transport mechanisms. Thus, toxicants that interfere with ATP production may cause distal regions to degenerate first. Agents that target tubulin also cause this type of injury because the tubulin-derived microtubules are critical for axonal transport. Augustus Waller, in the mid-nineteenth century,56 described the sequence of degenerative events that occurred following transection of a nerve fiber, effects subsequently known as Wallerian degeneration.54 The essential features of this type of degeneration include swelling of the axon in the proximal segment at the site of transection, dissolution and phagocytosis by inflammatory cells of the axon segment distal to the transection, and dissolution of myelin, with preservation and proliferation of Schwann cells along the length of the former axon. Certain neurotoxicants are capable of chemically transecting an axon, producing Wallerian degeneration similar to that occurring after transection. Axonopathy can manifest as sensory or motor function deficits or both. For most neurotoxicants, sensory changes are noticed first, followed by progressive involvement of motor neurons. OPIDN, organophosphateinduced delayed neuropathy, is historically important and has been much investigated. First described during Prohibition was an epidemic resulting from the consumption of “Ginger Jake,” alcoholic drinks deliberately contaminated with a triorthocresyl phosphate-containing oil in lieu of ginger extract. Within weeks of consuming the product, individuals experienced tingling and numbness in the hands and feet. If the dose was sufficient, this progressed to leg cramps, weakness of the limbs, and loss of coordination. Probably dose-related, some exposed individuals with minor symptoms improved, but many were permanently paralyzed. Since TOCP is an oil additive, episodes of OPIDN still occasionally occur, and it is also used as a model compound to study the delayed effects caused by some other organophosphate compounds such as leptophos. OPIDN does not appear to be related to the inhibition of acetylcholinesterase but rather to the inhibition of another neuronal esterase, the neuropathy target esterase or NTE.

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Neuronopathy. Neuronopathy refers to generalized damage to nerve cells, primary occurring at the nerve cell body. Many neurotoxicants produce their effects by promoting cell death in neurons. Excitotoxicity, or toxicity caused by glutamate and other excitatory amino acids, has been studied for its role in ischemic and seizure-induced brain damage. Domoic acid, a toxin produced by algae that frequently contaminate shellfish, binds to glutamate receptors and causes excitotoxic cell death (see Chapter 14). Functional toxic effects. Neurotoxicants may interfere with signaling processes within the nervous system by activating or inhibiting receptors, or by changing the amount of neurotransmitter available to activate receptors. This type of neurotoxicity is illustrated by the well-characterized actions of organophosphates and carbamates on acetylcholine signaling. Acetylcholinesterase inhibition. Organophosphates inhibit acetylcholinesterase, the enzyme that hydrolyzes acetylcholine, thus ending its receptor-stimulating activity. After acetylcholine has been released into the synapse or the neuromuscular junction, acetylcholinesterase terminates receptor-stimulating activity by its hydrolytic action. Many OPs found in human environments are not phosphates but thiophosphates, thionophosphates, thiophosphonates, etc., that are not effective inhibitors of acetylcholinesterase. All of these chemicals containing the P–S moiety are activated to phosphates or phosphonates by a CYP-dependent monooxygenase activity known as oxidative desulfuration. These biologically active oxons bind to the active site of acetylcholinesterase, covalently phosphorylating the serine residue in the catalytic site of the enzyme. The phosphorylation of acetylcholinesterase creates a relatively stable inactive enzyme that persists for hours to days before hydrolysis of the phosphate moiety occurs spontaneously, and acetylcholinesterase activity is restored. Carbamates also inhibit acetylcholinesterase by carbamylating the enzyme, although the stability of the carbamylated enzyme is much less than the phosphorylated enzyme and spontaneous reactivation occurs much faster than with organophosphates. Whereas organophosphates enhance neurotransmitter activity by inhibiting the breakdown of acetylcholine, many toxins produce hyperstimulation of receptors by directly binding and activating them (agonism). Others reduce receptor stimulation by prohibiting the neurotransmitter from activating them (antagonism). The Clostridium bacterial toxins, botulinum (causing botulism) and tetanospasmin (causing tetanus), block neurotransmission by inhibiting release of neurotransmitter into synapses and at motor end plates in muscle.

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6.2. Environmental neurotoxicants Environmental neurotoxicants are found in all human environments and in many cases represent significant human health problems. A small number of examples are shown in Table 4.1. It might be noted that the use of lead, once widely used as a gasoline and paint additive, as well as many other industrial and urban uses, is now largely banned in the USA and many other countries. Despite the dramatic drop in use, lead is still a major problem, particularly in the urban environment, due to paint residues from earlier use. Inorganic lead is known to cause direct damage to myelinating cells, and oligodendrocytes appear to be more sensitive to lead toxicity than astrocytes or neurons. The developmental effects of lead exposure may be a consequence of the preferential inhibition of oligodendrocyte precursor cell differentiation.

7. REPRODUCTIVE TOXICITY Reproductive toxicity results when a chemical has an adverse impact on the normal function and development of male or female reproductive tissues or reproductive behavior resulting in reduced fertility.57 The Table 4.1 Some examples of environmental neurotoxicants Neurotoxicant Toxic endpoint Environment(s)

Hexachlorophene

Demyelination

Domestic agroecosystem

Organotins

Demyelination

Occupational

Xylene

Demyelination

Occupational

Trichloroethylene

Demyelination

Occupational

Hexane

Axonopathy (Wallerian degeneration)

Occupational Urban (domestic)

Tri-o-cresyl phosphate

Axonopathy (OPIDN)

Occupational

Domoic acid

Neuronopathy

Occupational (fisheries) Urban (domestic)

Chlorpyrifos

Acetylcholinesterase inhibition

Agroecosystem urban

Carbaryl

Acetylcholinesterase inhibition

Agroecosystem urban

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reproductive system is controlled by the hypothalamic–pituitary–gonadal (HPG) axis via the release of hormones which act on target tissue containing specific hormone receptors, and chemicals may impact this system at multiple levels resulting in altered maturation or function. In adults, chemical exposure can impact normal sperm or egg development, fertilization, implantation, delivery, or lactation. During development, reproductive tissues are sensitive to chemicals, specifically testicular and ovarian tissues, and exposure may result in long-lasting effects including altered postnatal development, behavioral changes, or other changes that limit reproductive success.1 Exposure need only occur during specific windows of development to alter the organization of reproductive tissues and cause toxicity. Many endpoints may be used to determine if a chemical causes reproductive toxicity, and in vivo studies can assess directly sperm or egg development, mating and fertilization success, normal reproductive tissue development, embryo development, and birth success.58 Assessments can be made of external reproductive organs, the testes and ovaries, but also the accessory organs. In vitro receptor activity assays can determine if androgen, estrogen, progesterone, or other hormone receptors in hormone-responsive tissues are the target of reproductive toxicants, which can act as agonists or antagonists. Additionally, assays can be done to determine if a chemical inhibits hormone synthesis, by using, for example, rodent ex vivo testis or ovarian tissues.59,60 Another endpoint to be considered is whether a chemical alters the pathways that metabolize hormones. Diethylstilbestrol (DES) is the most widely recognized chemical causing reproductive toxicity. Administered to pregnant women until the 1970s, DES exposure in utero led to a host of reproductive problems in daughters and a higher incidence of a rare form of cervicovaginal cancer. DES was acting as an estrogen during development and is an example of a chemical that caused problems in adults after a fetal exposure. In the agroecosystem, a number of pesticides can cause reproductive toxicity. Though banned in the United States, dichlorodiphenyltrichloroethane (DDT) is still used in many parts of the world and acts as an estrogen agonist and androgen receptor antagonist. Other pesticides have been linked to reproductive toxicity including kepone, dibromochloropropane, and endosulfan.61,62 Triazole fungicides may alter normal testosterone homeostasis, affecting testosterone target tissues resulting in decreased fertility and altered xenobiotic and hormone metabolism in the liver.63–65 In the domestic environment, genistein and other isoflavones are endocrine disruptors found in plants, such as soybeans, which activate the

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estrogen receptor. Genistein has been shown to alter oocyte development in rodents, and the effects on human reproductive tissues are unclear. Metals found in the domestic and industrial environments, such as lead, can reduce sperm number and quality by interfering with the HPG axis, reduce testosterone levels, and disrupt development. Arsenic has the ability to alter the activity of progesterone and androgen receptors, and other metals found in industrial settings may have similar reproductive effects.66 A number of plastics are also found in the domestic environment in household items, including bisphenol A and phthalates. Phthalate exposure during development has been shown to cause testicular dysgenesis syndrome, consisting of undescended testes, hypospadia, decreased anogenital distance, and other disruptions in testicular organization function in rodents. Some studies suggest that a decrease in anogenital distance may also be the case in humans.67 In adults, phthalate levels have been correlated with altered semen quality.68,69 Phthalates and bisphenol A can also impact ovarian function.70 Exposure to aryl hydrocarbon receptor agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or other polycyclic aromatic hydrocarbons in deployment-related and industrial environments have been shown to affect reproductive tissues via multiple mechanisms.71,72

8. ENDOCRINE TOXICITY Endocrine systems function to maintain control over many of the other systems of the body via glands that release hormones that circulate in the blood stream. Hormones act on target tissues and cells that respond to hormones via various signal transduction pathways, such as receptors. The endocrine system consists of many different glands that secrete hormones including, for example, the hypothalamus, pituitary, thyroid, adrenal, ovaries, and testes. Endocrine toxicity results when a chemical interferes with the synthesis, secretion, transport, metabolism, binding action, or elimination of hormones necessary for endocrine functions resulting in loss of normal tissue function, development, growth, or reproduction.73,74 Endocrine signaling occurs in feedback mechanism in which the central nervous system initiates the signaling via the hypothalamus. Hormones produced by the hypothalamus act on the pituitary, which secretes additional hormones which act on the gonads to produce androgens or estrogens, the thyroid gland to produce thyroid hormones, or the adrenal glands to produce corticosteroids. The hormones produced act on the hypothalamus in a negative manner to limit further hormone production of target organs. Hormonal signaling acts by hormone binding to specific

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nuclear receptor transcription factors such as the androgen receptor, estrogen receptor, thyroid receptor (TR), or glucocorticoid receptor found in target tissues. Forty-eight receptors have been identified in humans, and once a receptor is activated by a hormone, it alters the expression of specific genes, resulting in altered protein levels resulting in the hormone’s change in cellular function. Confounding the study of chemicals that affect endocrine systems is that some endocrine-disrupting chemicals cause nonmonotonic dose responses, suggesting that low doses of EDCs may have significant unanticipated impacts on some endpoints.75 Endpoints then can be measured in assessing if a chemical causes endocrine toxicity includes receptor activity assays. Receptors in hormone-responsive tissues can be the target of environmental chemicals, which can alter the ability of hormones to bind and activate their specific receptors leading to disruption of the normal signaling cascade. Chemicals may be receptor agonists, which activate the receptor like the hormone that normally binds to the receptor. Alternatively, chemicals may be receptor antagonists, where the receptor is bound by the chemical and receptor activity is inhibited. The endocrine system can also be impacted by chemicals which inhibit hormone synthesis or if hormone clearance is enhanced by increased expression of xenobioticmetabolizing enzymes. Disruption of the endocrine system during development can affect the normal developmental processes. The drug diethylstilbestrol (DES), which was administered to pregnant women from the 1940s to the early 1970s, is an example of a chemical that impacts the endocrine system and disrupts normal development. The drug DES has an activity that is similar to that of hormone estrogen, and DES exposure during developmental windows, where estrogen should not be present or at low levels, resulted in altered development of the reproductive system. The consequences of DES exposure were often not seen until puberty. In the agricultural environment exposure to fungicides, such as vinclozolin and triazole impact androgen and thyroid pathways. In the agroecosystems, also, the pesticides endosulfan, chlorpyrifos, methoxychlor, and DDT have been linked to endocrine toxicity. Pesticides have been linked with disruption of many endocrine pathways, such as androgen and thyroid signaling.62,73,76 Disruption of thyroid hormone (TH) signaling can occur by an environmental chemical altering iodine uptake at the thyroid, inhibition of enzymes required for thyroid hormone synthesis, or enhanced TH excretion.77 The environmental contaminants known as polychlorinated biphenyls (PCBs) are able to affect thyroid hormone function with some PCBs acting

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as agonists for the thyroid hormone at the TR. In the domestic and occupational environments, the plastic bisphenol A, the polybrominated biphenyls, and the flame retardants polybrominated diphenyl ethers are thought to alter TH functions.78 Other pathways in the endocrine system may also be targeted by endocrine toxic compounds, for example, endocrine signaling leads to adipogenesis, which is regulated by peroxisome proliferator-activated receptor (PPAR) pathway. PPAR may be activated by organotins that are used in many industrial settings and as marine antifouling agents.78 In the domestic environment, the metal arsenic (As) has the ability to alter glucocorticoid receptor activity, as well as the mineralocorticoid, progesterone, and androgen receptors, by a mechanism involving receptor coactivator interactions.66 A number of endocrine-disrupting plastics are also found in the domestic environment, including bisphenol A and phthalates. Phthalates affect reproductive development but also inhibit the enzymatic activity of 11b-hydroxysteroid dehydrogenase, which is responsible for the inactivation of endogenous glucocorticoids.79 Concern of the impact of environmental chemicals on the endocrine led to the US Environmental Protection Agency to develop an Endocrine Disruptor Screening Program (EDSP) consisting of multiple types of assays. The first phase of testing to determine whether a chemical has an effect on endocrine systems included receptor binding assays, receptor activity assays, hormone synthesis assays, assessment of impact on reproductive tissues in male and female rats, frog metamorphosis assay, and fish life cycle assay.80

9. IMMUNOTOXICITY: RESPIRATORY 9.1. The immune system and immunotoxic endpoints The immune system is a complex and highly cooperative system of cells, tissues, and organs whose primary function is to protect an organism from infection by foreign organisms and from newly arising neoplasms. These tasks can be accomplished in a nonspecific manner, such as the ingestion of particles by phagocytes, or in a very specific manner, such as the neutralization of some bacterial endotoxins by antibodies, or in ways that have both specific and nonspecific components, such as antibody-dependent cellular cytotoxicity, where the binding of specific antibodies enables nonspecific phagocytes to destroy the cells. Most organisms have some form of nonspecific defense, but only vertebrates have the capability to make a specific, adaptive, anamnestic response. This is primarily due to the B and T cells. A brief

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Totipotent stem cell

Killer cell (LGL)

Natural Killer cell

Monocyte

Macrophage

Puripotent stem cell

Lymphoid precursor

Null cell

CFU-GM

Myeloid precursor (CFU-GEMM)

Megakaryocyte

Erythyroid progenitor

Neutrophil Mast cell

Basophil Esosinophil

Figure 4.1 Development and cells of the immune system. Modified from Klassen CD, editors. Casarett and Doull's toxicology: the basic sciences of poisons. 7th ed. New York: MaGraw-Hill Companies 81.

summary of the interactions and functions of the immune system’s cells is shown in Fig. 4.1. All of these cells, as well as the erythrocytes, are derived from the pluripotent stem cell in the bone marrow. Progenitors of the lymphocytes migrate to the primary lymphoid organs, the bone marrow, and the thymus, where they mature into B and T cells, respectively. From here, the lymphocytes enter the circulation and home to the secondary lymphoid organs. The lymphocytes are continually circulated through these organs, via the blood stream and lymphatic system, which serve as the major filtering organs of the lymph (lymph nodes), blood (spleen), gut (Peyer’s patches, appendix) and upper respiratory tract (adenoids, tonsils). Nonspecific responses to foreign material are generally initiated by phagocytic cells at the site of infection or irritation. Also, some microorganisms trigger the alternative pathway of complement activation. If this response is inadequate, the antigen load increases, and the lymphocyte response is activated. It is in the lymph nodes and spleen that the humoral responses to blood- and lymph-borne antigens are initiated. The humoral immune response consists of those interactions that lead to the production of circulating antibodies. These

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antibodies can have a number of different roles in host defense. They can neutralize toxins by binding to the active site and can prevent mucosal attachment of gut parasites by a similar mechanism. Additionally, antibodies bound to a cell surface can activate the complement pathways, inducing inflammation, and can enhance the phagocytic efficiency of macrophages. Since antibodies are multivalent, they can also agglutinate viruses and bacteria into more easily removed particles. Cell-mediated immunity, the other arm of the immune response, leads to the generation of cytotoxic T cells. Cytotoxic T cells are able to destroy virally infected cells, tumor cells, and foreign tissue. Typically, extracellular bacteria and viruses induce humoral immunity, whereas fungi, intracellular viruses, cancer, and foreign tissue induce cell-mediated immunity, although this is by no means absolute.81 Environmental agents and other xenobiotics such as clinical drugs and drugs of abuse can affect the immune system in several ways. They can cause immunosuppression, either by a general decrease in cellularity or by a decrease in the numbers and/or function of particular cell types, or cause uncontrolled proliferation. Both of these can lead to substantial alterations in host defense mechanisms and therefore to increased vulnerability to pathogens and neoplasms. Additionally, environmental agents can cause the immune system to respond in a way that is detrimental to the host, as in allergic responses and autoimmunity. Clearly, exposure to xenobiotics can have a number of effects on the immune system that in turn can affect an array of health outcomes. In some areas of immunotoxicology, significant progress has been made in terms of identifying and understanding the risks associated with xenobiotic exposure. In other areas, more research is needed. Two useful general treatments by Selgrade82,83 provide further information on the immune system.

9.2. Immunotoxicants The immune system is the body’s primary defense against infectious agents and, in some cases, from the cells of newly arising neoplasms. As a consequence, individuals with immune deficiencies resulting from genetic defects, diseases, or drug therapies are likely to be more susceptible to infections or to certain types of cancer, either of which could be lifethreatening. On the other hand, the immune system may react to foreign substances that would otherwise be relatively innocuous, such as certain chemicals, pollens, or house dust with resulting allergic reactions that can include pathologies ranging from skin rashes and rhinitis to more lifethreatening asthmatic and anaphylactic reactions. A crucial part of immune

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function is the ability to distinguish endogenous components (“self”) from potentially harmful exogenous components (“nonself”). Failure to make this distinction in an immune system with impaired function can result in autoimmune disease. Immunotoxicology is the study of undesired effects resulting from the interactions of xenobiotics with the immune system.82,83 Although many of the interactions of the immune system involve complex natural products such as pollen grains, bacteria, and viruses, xenobiotics are known to impact the system causing immunosuppression, allergic disease, and autoimmune disease. Immunosuppression. Many drugs, particularly those used in cancer therapy and in the control of inflammation, can produce alterations in the immune system that lead to impaired immune function. They will not be discussed further. However, environmental agents can also depress immune function. Benzene is a potent bone marrow toxicant causing overall decreases in immune cell numbers, as well as alterations in function. Polychlorinated biphenyls, polybrominated biphenyls, and some metals are among the compounds that can cause immunosuppression at sublethal doses. Depression of immune function can lead to increased susceptibility to bacterial, viral, and parasitic infections and possibly increased incidence of neoplasms.84 Allergic disease. Xenobiotics can affect allergic disease in one of the two ways. They can themselves act as antigens and elicit hypersensitivity (i.e., allergic) responses, or they can enhance the development or expression of allergic responses to commonly encountered allergens, such as dust mites. Chemicals that act as allergens include certain proteins that can by themselves induce an immune response and low-molecular-weight chemicals (known as haptens) that are too small to induce a specific immune response but may react with a protein to induce an immune response that is then hapten-specific. Haptens have been associated with both allergic contact dermatitis, sometimes called contact hypersensitivity, and respiratory hypersensitivity. Systemic hypersensitivity, the most extreme manifestation of which is anaphylaxis, can also occur in response to low-molecular-weight compounds. Contact dermatitis, also referred to as contact hypersensitivity, is a common immunotoxic occupational health problem. It is often seen in occupational groups such as metal workers, hair dressers, and food-processing workers and often affects the hands.85,86 Respiratory hypersensitivity. Both proteins and small xenobiotic molecules can cause or exacerbate respiratory allergies, the most common manifestation of which is asthma. While proteins are the most common cause, highly reactive xenobiotics such as toluene diisocyanate are also respiratory

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allergens, probably mediated through their ability to form protein adducts, the entity that is then seen as “nonself” and elicits the allergic response.87 Other chemicals encountered in the industrial environment include metals, paints, and plastics. A number of molds, plants, and animal byproducts in the agricultural environment can also trigger respiratory hypersensitivity.88 Autoimmune Disease. Autoimmune diseases result from a breakdown of immunological tolerance leading to immune responses against self-molecules that involve activation of both innate and adaptive immune responses. Autoimmune disorders can affect virtually any site in the body and present as a spectrum of diseases. Autoimmune diseases affect about 3% of the population and comprise a diverse array of both organ-specific (e.g., type 1 diabetes, thyroiditis) and systemic (systemic lupus erythematosus) diseases. Epidemiologic studies suggest associations with specific genetic loci and environmental factors, including exposures to certain drugs, chemicals, and infectious agents. In many cases, women appear to be more vulnerable than men. Xenobiotics have the potential to affect the development, progression, or severity of autoimmune disease. In the industrial environment, exposure to trichloroethylene, metals, asbestos, and silica has been linked to autoimmune diseases.89–92 A variety of mechanisms could contribute to xenobiotic effects on the development and maintenance of immune tolerance or unmasking or modification of self-proteins. There is also evidence that exposure to certain drugs and endocrine disruptors are a concern in this regard. Developmental effects. Finally, there is growing concern that the developing immune system may be vulnerable to xenobiotic exposures and that perinatal and/or in utero exposures may have a lifelong impact on susceptibility to infectious, allergic, or autoimmune disease. Chemicals of concern include benzo(a)pyrene, TCDD, the metals mercury and lead, and some pesticides.93,94

10. CONCLUDING REMARKS Human exposure to chemicals will continue to be a concern in the many environments we occupy, ranging from occupational to domestic settings. Efforts such as the National Health and Nutrition Examination Survey (NHANES), conducted by the Centers for Disease Control and Prevention (CDC), are measuring chemicals or their metabolites in blood, serum, and urine samples from a large number of people to identify environmental chemical exposure levels in the US population. Epidemiological studies, for example, the

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Agricultural Health Study (AHS), are attempting to link environmental chemical exposures with human disease. These approaches and the prospective use of methods to identify chemicals that pose potential hazards, including quantitative structure–activity relationships (QSAR), HTS, and systems biology approaches, will attempt to prioritize chemicals for further study and identify the potential hazards of new and emerging chemicals. These and other methods will aid in the identification of new toxic endpoints, at early time points in the process of toxicity, to inform future regulatory decisions.

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