a textbook of modern toxicology phần 6 pdf

Whenmore 3,4-epoxide is produced than can readily be detoxified, cell injury increases.Pretreatment of animals with inhibitors of cytochrome P450 is known to decrease tis-sue necrosis by

accumulation of fibrous material causes severe restriction in blood flow and in theliver’s normal metabolic and detoxication processes This situation can in turn causefurther damage and eventually lead to liver failure In humans, chronic use of ethanol

is the single most important cause of cirrhosis, although there is some dispute as towhether the effect is due to ethanol alone or is also related to the nutritional deficienciesthat usually accompany alcoholism

14.3.7 Oxidative Stress

Oxidative stress has been defined as an imbalance between the prooxidant/antioxidantsteady state in the cell, with the excess of prooxidants being available to interact withcellular macromolecules to cause damage to the cell, often resulting in cell death.Although the occurrence of reactive oxygen species in normal metabolism and theconcept of oxidative stress was derived from these studies, it is apparent that oxidativestress can occur in almost any tissue, producing a variety of deleterious effects To date,

a number of liver diseases, including alcoholic liver disease, metal storage diseases,and cholestatic liver disease, have been shown to have an oxidative stress component.Reactive oxygen and reactive nitrogen radicals can be formed in a number of ways(Figure 14.2), the former primarily as a by-product of mitochondrial electron transport.Superoxide, hydrogen peroxide, singlet oxygen, and hydroxyl can all arise from thissource Other sources include monooxygenases and peroxisomes If not detoxified,reactive oxygen species can interact with biological macromolecules such as DNAand protein or with lipids Once lipid peroxidation of unsaturated fatty acids in phos-pholipids is initiated, it is propagated in such a way as to have a major damagingeffect on cellular membranes The formation, detoxication by superoxide dismutaseand by glutathione-dependent mechanisms, and interaction at sites of toxic action areillustrated in Figure 14.2

14.3.8 Carcinogenesis

The most common type of primary liver tumor is hepatocellular carcinoma; other typesinclude cholangiocarcinoma, angiosarcoma, glandular carcinoma, and undifferentiatedliver cell carcinoma Although a wide variety of chemicals are known to induce livercancer in laboratory animals (Table 14.1), the incidence of primary liver cancer inhumans in the United States is very low

Some naturally occurring liver carcinogens are aflatoxin, cycasin, and safrole Anumber of synthetic chemicals have been shown to cause liver cancer in animals,including the dialkylnitrosamines, dimethylbenzanthracene, aromatic amines such as

Sites of blocking oxidant challenges by antioxidant defenses.

electron transport proteins activated phagocytes redox cycling

NO nitric oxide synthetase

metal ions

GSH redox cycle

biomolecules (DNA, lipid, protein)

Figure 14.2 Molecular targets of oxidative injury (From D J., Reed, Introduction to ical Toxicology, 3rd ed., Wiley, 2001.)

Biochem-2-naphthylamine and acetylaminofluorene, and vinyl chloride The structure and vation of these compounds can be found in Chapters 7 and 8 In humans, the mostnoted case of occupation-related liver cancer is the development of angiosarcoma, a raremalignancy of blood vessels, among workers exposed to high levels of vinyl chloride

acti-in manufacturacti-ing plants For a discussion of chemical carcacti-inogenesis, see Chapter 12

14.4 MECHANISMS OF HEPATOTOXICITY

Chemically induced cell injury can be thought of as involving a series of events ring in the affected animal and often in the target organ itself:

occur-ž The chemical agent is activated to form the initiating toxic agent

ž The initiating toxic agent is either detoxified or causes molecular changes inthe cell

ž The cell recovers or there are irreversible changes

ž Irreversible changes may culminate in cell death

Cell injury can be initiated by a number of mechanisms, such as inhibition ofenzymes, depletion of cofactors or metabolites, depletion of energy (ATP) stores, inter-action with receptors, and alteration of cell membranes In recent years attention hasfocused on the role of biotransformation of chemicals to highly reactive metabolitesthat initiate cellular toxicity Many compounds, including clinically useful drugs, cancause cellular damage through metabolic activation of the chemical to highly reactivecompounds, such as free radicals, carbenes, and nitrenes (Chapters 7 and 8).

These reactive metabolites can bind covalently to cellular macromolecules such asnucleic acids, proteins, cofactors, lipids, and polysaccharides, thereby changing theirbiologic properties The liver is particularly vulnerable to toxicity produced by reac-tive metabolites because it is the major site of xenobiotic metabolism Most activationreactions are catalyzed by the cytochrome P450 enzymes, and agents that induce theseenzymes, such as phenobarbital and 3-methylcholanthrene, often increase toxicity Con-versely, inhibitors of cytochrome P450, such as SKF-525A and piperonyl butoxide,frequently decrease toxicity

Mechanisms such as conjugation of the reactive chemical with glutathione are tective mechanisms that exist within the cell for the rapid removal and inactivation ofmany potentially toxic compounds Because of these interactions, cellular toxicity is afunction of the balance between the rate of formation of reactive metabolites and therate of their removal Examples of these interactions are presented in the followingdiscussions of specific hepatotoxicants

Typically free radicals may participate in a number of events (Figure 14.4), such

as covalent binding to lipids, proteins, or nucleotides as well as lipid peroxidation It

C

Cl

Cl Cl

low O 2

Binding to lipids, Lipid peroxidation

Figure 14.3 Metabolism of carbon tetrachloride and formation of reactive metabolites (From

P E Levi, A Textbook of Modern Toxicology, 2nd ed., Appleton and Lange, 1997.)

Free Radicals

Protein binding DNA binding

SH oxidation Depletion of cofactors Lipid peroxidation

Figure 14.4 Summary of some toxic effects of free radicals (From P E Levi, A Textbook of Modern Toxicology, 2nd ed., Appleton and Lange, 1997.)

Figure 14.5 Schematic illustrating lipid peroxidation and destruction of membranes (From

P E Levi, A Textbook of Modern Toxicology, 2nd ed., Appleton and Lange, 1997.)

is now thought that CCl3ž, which forms relatively stable adducts, is responsible forcovalent binding to macromolecules, and the more reactive CCl3O2ž, which is formedwhen CCl3ž reacts with oxygen, is the prime initiator of lipid peroxidation

Lipid peroxidation (Figure 14.5) is the initiating reaction in a cascade of events,starting with the oxidation of unsaturated fatty acids to form lipid hydroperoxides,which then break down to yield a variety of end products, mainly aldehydes, whichcan go on to produce toxicity in distal tissues For this reason cellular damage resultsnot only from the breakdown of membranes such as those of the endoplasmic reticulum,mitochondria, and lysosomes but also from the production of reactive aldehydes thatcan travel to other tissues It is now thought that many types of tissue injury, includinginflammation, may involve lipid peroxidation

14.5.2 Ethanol

Alcohol-related liver diseases are complex, and ethanol has been shown to interact with

a large number of molecular targets Ethanol can interfere with hepatic lipid metabolism

in a number of ways and is known to induce both inflammation and necrosis in theliver Ethanol increases the formation of superoxide by Kupffer cells thus implicatingoxidative stress in ethanol-induced liver disease Similarly prooxidants (reactive oxygenspecies) are produced in the hepatocytes by partial reactions in the action of CYP2E1,

an ethanol-induced CYP isoform The formation of protein adducts in the microtubules

by acetaldehyde, the metabolic product formed from ethanol by alcohol dehydrogenase,plays a role in the impairment of VLDL secretion associated with ethanol

14.5.3 Bromobenzene

Bromobenzene is a toxic industrial solvent that is known to produce centrilobularhepatic necrosis through the formation of reactive epoxides Figure 14.6 summarizes

O

O

Br Br

Br

OH OH

Br OH

Br

OH 2-Bromophenol

to form the nontoxic 2-bromophenol The more stable 3,4-epoxide is the form mostresponsible for covalent binding to cellular proteins A number of pathways exist fordetoxication of the 3,4-epoxide: rearrangement to the 4-bromophenol, hydration to the3,4-dihydrodiol catalyzed by epoxide hydrolase, or conjugation with glutathione Whenmore 3,4-epoxide is produced than can readily be detoxified, cell injury increases.Pretreatment of animals with inhibitors of cytochrome P450 is known to decrease tis-sue necrosis by slowing down the rate of formation of the reactive metabolite, whereaspretreatment of animals with certain P450 inducers can increase the toxicity of bro-mobenzene, (e.g., the P450 inducer phenobarbital increases hepatotoxicity by inducting

a P450 isozyme that preferentially forms the 3,4-epoxide) However, pretreatment withanother P450 inducer, 3-methylcholanthrene, decreases bromobenzene hepatotoxicity

by inducing a form of P450 that produces primarily the less toxic 2,3-epoxide

levels, however, resulting in extensive covalent binding of the reactive metabolite toliver macromolecules with subsequent hepatic necrosis The early administration of

sulfhydryl compounds such as cysteamine, methionine, and N -acetylcysteine is very

effective in preventing liver damage, renal failure, and death that would otherwisefollow an acetaminophen overdose These agents are thought to act primarily by stim-ulating glutathione synthesis

In laboratory animals the formation of the acetaminophen-reactive metabolite, theextent of covalent binding, and the severity of hepatotoxicity can be influenced byaltering the activity of various P450 isozymes Induction of P450 isozymes with phe-nobarbital, 3-methylcholanthrene, or ethanol increases toxicity, whereas inhibition ofP450 with piperonyl butoxide, cobalt chloride, or metyrapone decreases toxicity Con-sistent with these effects in animals, it appears that the severity of liver damage afteracetaminophen overdose is greater in chronic alcoholics and patients taking drugs thatinduce the levels of the P450 isozymes responsible for the activation of acetaminophen

14.6 METABOLIC ACTIVATION OF HEPATOTOXICANTS

Studies of liver toxicity caused by bromobenzene, acetaminophen, and other pounds have led to some important observations concerning tissue damage:

com-ž Toxicity may be correlated with the formation of a minor but highly reactiveintermediate

ž A threshold tissue concentration of the reactive metabolite must be attained beforetissue injury occurs

ž Endogenous substances, such as glutathione, play an essential role in protectingthe cell from injury by removing chemically reactive intermediates and by keepingthe sulfhydryl groups of proteins in the reduced state

ž Pathways such as those catalyzed by glutathione transferase and epoxide lases play an important role in protecting the cell

hydro-ž Agents that selectively induce or inhibit the xenobiotic metabolizing enzymesmay alter the toxicity of xenobiotic chemicals

These same principles are applicable to the toxicity caused by reactive metabolites

in other organs, such as kidney and lung as will be illustrated in the following sections

SUGGESTED READING

Hodgson, E., and S A Meyer Pesticides In Comprehensive Toxicology: Hepatic and testinal Toxicology, vol 9, I G Sipes, C A McQueen, and A J Gandolfi, eds New York:

Gastroin-Elsevier Science, 1997, p 369.

Meyer, S A Hepatotoxicity In An Introduction to Biochemical Toxicology, 3rd ed., E Hodgson

and R C Smart, eds New York: Wiley, 2001, p 487.

Reed, D J Mechanisms of chemically induced cell injury and cellular protection mechanisms.

In An Introduction to Biochemical Toxicology, 3rd ed., E Hodgson and R C Smart, eds New

York: Wiley, 2001, p 221.

Treinen-Moslen, M Toxic responses of the liver In Casarett and Doull’s Toxicology: The Basic Sciences of Poisons, 6th ed., C D Klaassen, ed New York: McGraw-Hill, 2001, p 471.

ERNEST HODGSON and PATRICIA E LEVI

15.1 INTRODUCTION

15.1.1 Structure of the Renal System

The renal system consists of the kidneys and their vasculature and innervation, thekidneys each draining through a ureter into a single median urinary bladder, and thelatter draining to the exterior via a single duct, the urethra The kidney has three majoranatomical areas: the cortex, the medulla, and the papilla

The renal cortex is the outermost region of the kidney and contains glomeruli,proximal and distal tubules, and peritubular capillaries Cortical blood flow is high,the cortex receiving approximately 90% of the renal blood flow Since blood-bornetoxicants will be delivered preferentially to the cortex, they are more likely to affectcortical functions rather than those of medulla or papilla The renal medulla is themiddle portion and contains primarily loops of Henle, vasa recta, and collecting ducts.Although the medulla receives only about 6% of the renal blood flow, it may beexposed to high concentrations of toxicants within tubular structures The papilla isthe smallest anatomical portion of the kidney and receives only about 1% of the renalblood flow Nevertheless, because the tubular fluid is maximally concentrated andluminal fluid is maximally reduced, the concentrations of potential toxicants in thepapilla my be extremely high, leading to cellular injury in the papillary tubular and/orinterstitial cells

The nephron is the functional unit of the kidney It is described in detail in Chapter 10and illustrated in Figure 10.1

15.1.2 Function of the Renal System

The primary function of the renal system is the elimination of waste products, derivedeither from endogenous metabolism or from the metabolism of xenobiotics The lat-ter function is discussed in detail in Chapter 10 The kidney also plays an importantrole in regulation of body homeostasis, regulating extracellular fluid volume, and elec-trolyte balance

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Other functions of the kidney include the synthesis of hormones that affect bolism For example, 25-hydroxy-vitamin D3 is metabolized to the active form, 1,25-dihydroxy-vitamin D3 Renin, a hormone involved in the formation of angiotensinand aldosterone, is formed in the kidney as are several prostaglandins While kidneytoxicity could affect any of these functions, the effects used clinically to diagnosekidney damage are related to excretory function damage, such as increases in urinaryglucose, amino acids, or protein, changes in urine volume, osmolarity, or pH Similarlychanges in blood urea nitrogen (BUN), plasma creatinine, and serum enzymes can beindicative of kidney damage.

meta-In animal studies of nephrotoxicity not only can histopathology be carried out butvarious biochemical parameters can be compared with those from untreated animals.They include lipid peroxidation and covalent binding to tissue macromolecules

15.2 SUSCEPTIBILITY OF THE RENAL SYSTEM

Several factors are involved in the sensitivity of the kidney to a number of toxicants(Table 15.1), although the high renal blood flow and the increased concentration ofexcretory products following reabsorption of water from the tubular fluid are clearly

of major importance Although the kidneys comprise less than 1% of the body mass,they receive around 25% of the cardiac output Thus significant amounts of exogenouschemicals and/or their metabolites are delivered to the kidney

A second important factor affecting the kidneys sensitivity to chemicals is its ability

to concentrate the tubular fluid and, as a consequence, as water and salts are removed,

to concentrate any chemicals it contains Thus a nontoxic concentration in the plasmamay be converted to one that is toxic in the tubular fluid The transport characteristics

of the renal tubules also contribute to the delivery of potentially toxic concentrations

of chemicals to the cells If a chemical is actively secreted from the blood into thetubular fluid, it will accumulate initially within the cells of the proximal tubule or,

if it is reabsorbed from the tubular fluid, it will pass into the cells in relatively highconcentration

The biotransformation of chemicals to reactive, and thus potentially toxic, lites is a key feature of nephrotoxicity Many of the same activation reactions found

metabo-in the liver are also found metabo-in the kidney and many toxicants can be activated metabo-ineither organ, including acetaminophen, bromobenzene, chloroform, and carbon tetra-chloride, thus having potential for either hepatotoxicity or nephrotoxicity Some regions

of the kidney have considerable levels of xenobiotic metabolizing enzymes, larly cytochrome P450 in the pars recta of the proximal tubule, a region particularlysusceptible to chemical damage Since reactive metabolites are generally unstable, andtherefore more or less transient, they are likely to interact with cellular macromolecularclose to the site of generation Thus, although the activity of activation enzymes such as

particu-Table 15.1 Factors Affecting the Susceptibility of the Kidney to Toxicants

High renal blood flow

Concentration of chemicals in tubular fluid

Reabsorption and/or secretion of chemicals through tubular cells

Activation of protoxicants to reactive, and potentially toxic, metabolites

cytochrome P450 is lower in the kidney that in the liver, they are of greater importance

in nephrotoxicity than those of the liver due to their proximity to site of action

As with toxicity in other organs the ultimate expression of a toxic end point is theresult of a balance between the generation of reactive metabolites and their detoxication.The high levels of glutathione found in the kidney doubtless play an important role inthe detoxication process

15.3 EXAMPLES OF NEPHROTOXICANTS

15.3.1 Metals

Many heavy metals are potent nephrotoxicants, and relatively low doses can duce toxicity characterized by glucosuria, aminoaciduria, and polyuria As the doseincreases, renal necrosis, anuria, increased BUN, and death will occur Several mecha-nisms operate to protect the kidney from heavy metal toxicity After low dose exposureand often before detectable signs of developing nephrotoxicity, significant concentra-tions of metal are found bound to renal lysosomes This incorporation of metals intolysosomes may result from one or more of several mechanisms, including lysoso-mal endocytosis of metal-protein complexes, autophagy of metal-damaged organellessuch as mitochondria, or binding of metals to lipoproteins within the lysosome Expo-sure to high concentrations, however, may overwhelm these mechanisms, resulting intissue damage

exposure to cadmium dust In Japan, a disease called Itai-itai Byo is known to occuramong women who eat rice grown in soils with a very high cadmium content Thedisease is characterized by anemia, damage to proximal tubules, and severe bone andmineral loss Cadmium is excreted in the urine mainly as a complex (CdMT) withthe protein metallothionein (MT) MT is a low molecular weight protein synthesized

in the liver It contains a large number of sulfhydryl groups that bind certain metals,including cadmium The binding of cadmium by MT appears to protect some organssuch as the testes from cadmium toxicity At the same time, however, the complexmay enhance kidney toxicity because the complex is taken up more readily by thekidney than is the free metal ion Once inside the cell, it is thought that the cadmium

is released, presumably by decomposition of the complex within the lysosomes.Cadmium has a long biological half-life, 10 to 12 years in humans; thus low-levelchronic exposure will eventually result in accumulation to toxic concentrations

Lead Lead, as Pb2+, is taken up readily by proximal tubule cells, where it damagesmitochondria and inhibits mitochondrial function, altering the normal absorptive func-tions of the cell Complexes of lead with acidic proteins appear as inclusion bodies

in the nuclei of tubular epithelium cells These bodies, formed before signs of leadtoxicity occur, appear to serve as a protective mechanism

proximal tubule cell In low concentrations, mercury binds to the sulfhydryl groups ofmembrane proteins and acts as a diuretic by inhibiting sodium reabsorption Organomer-curial diuretics were introduced into clinical practice in the 1920s and were used

clinically into the 1960s Despite their widespread acceptance as effective therapeuticdiuretics, it was well known that problems related to severe kidney toxicity were pos-sible However, in the absence of other effective drugs, the organomercurials proved

to be effective, sometimes life-saving, therapeutic agents More recently rial chemicals have been implicated as environmental pollutants, responsible for renaldamage in humans and animals

which is filtered by the glomerulus As a result of acidification in the proximal tubule,the bicarbonate complex dissociates, followed by reabsorption of the bicarbonate ion;the released UO2 + then becomes attached to the membrane of the proximal tubule

cells The resultant loss of cell function is evidenced by increased concentrations ofglucose, amino acids, and proteins in the urine

15.3.2 Aminoglycosides

Certain antibiotics, most notably the aminoglycosides, are known to be nephrotoxic

in humans, especially in high doses or after prolonged therapy The group of otics includes streptomycin, neomycin, kanamycin, and gentamycin Aminoglycosidesare polar cations that are filtered by the glomerulus and excreted unchanged intothe urine In the proximal tubule, the aminoglycosides are reabsorbed by binding toanionic membrane phospholipids, followed by endocytosis and sequestration in lyso-somes (Figure 15.1) It is thought that when a threshold concentration is reached, thelysosomes rupture, releasing hydrolytic enzymes that cause tissue necrosis

antibi-15.3.3 Amphotericin B

With some drugs, renal damage may be related to the drugs’ biochemical mechanism

of action For example, the polymycins, such as amphotericin B, are surface-activeagents that bind to membrane phospholipids, disrupting the integrity of the membraneand resulting in leaky cells

L

AG

AG

AG AG

AG

AG M

Figure 15.1 Possible cellular interactions of aminoglycosides AG = aminoglycoside; M = mitochondrion; L= lysosome (From E Hodgson and P E Levi, eds., A Textbook of Modern Toxicology 2nd ed., Appleton and Lange, Stamford, CT, 1997.)

15.3.4 Chloroform

Chloroform is a common industrial organic solvent that can be a hepatotoxicant or

a nephrotoxicant in both humans and animals As a nephrotoxicant it is both speciesand gender dependent For example, following chloroform administration male micedevelop primarily kidney necrosis whereas female develop liver necrosis

As a nephrotoxicant, chloroform most probably undergoes metabolic activation inthe kidney itself Chloroform is metabolized to phosgene (Figure 15.2) by a cytochromeP450-dependent reaction, probably proceeding via an unstable hydroxylated product,trichloromethanol Phosgene is capable of binding to cellular proteins to produce thecellular necrosis associated with chloroform toxicity to the kidney Phosgene canalso be further metabolized by a number of reactions (Figure 15.2), and as withmost chemical-induced toxicity, the final expression of toxicity depends on a balancebetween activation and detoxication

15.3.5 Hexachlorobutadiene

Hexachlorobutadiene is an industrial solvent and heat-transfer agent It is a widespreadenvironmental contaminant that is a potent and relatively specific nephrotoxicant Hex-achlorobutadiene first forms a glutathione conjugate, which is further metabolized bythe mercapturic acid pathway to a cysteine conjugate (see Chapter 7 for details ofglutathione conjugation and the mercapturic acid pathway) In the kidney, the cys-teine conjugate is cleaved to a reactive intermediate by the enzyme, cysteine conjugate

β-lyase

Cl C

H Cl Cl

Cytochrome P-450,

O 2 , microsomes Cl C

OH Cl Cl

Cl C

O Cl

H C S

C

O H

N

OH

C O H

Tox-Toxicology of the Nervous System

BONITA L BLAKE

16.1 INTRODUCTION

Many substances alter the normal activity of the nervous system Sometimes theseeffects are immediate and transient, like the stimulatory effect of a cup of coffee or aheadache from the fresh paint in your office Other effects can be much more insidious,like the movement disorders suffered by miners after years of chronic manganese intox-ication Many agents are safe or even therapeutic at lower doses but become neurotoxic

at higher levels Trace metals and pyridoxine (vitamin B-6) fall into this category ofdose-dependent effects Since these agents affirm the maxim, “the dose makes thepoison,” it becomes necessary to have a meaningful definition of nervous system poi-

soning, or neurotoxicity Neurotoxicity refers to the ability of an agent to adversely affect the structural or functional integrity of the nervous system Structural damage to

nervous system components usually results in altered functioning, although the reverse

is not always true Alterations in nervous system function may occur through toxicantinteractions with the normal signaling mechanisms of neurotransmission, resulting inlittle or no structural damage Nevertheless, it is easier to identify alterations, be theystructural or functional, than it is to define adversity For example, the stimulant effect

of a morning cup of coffee may be too anxiety provoking for some individuals but anecessity to others

In this chapter a brief introduction to the nervous system is presented and its tions are described A discussion of some of the mechanisms of structural and functionalneurotoxicant effects follows These descriptions are not exhaustive, they are meant toillustrate the concepts of toxicant interaction with the nervous system Finally somemethods for testing toxicant effects in the nervous system are explored

func-16.2 THE NERVOUS SYSTEM

Most multicellular animals possess a nervous system In every case the function ofthe nervous system is to receive information about the external and internal envi-ronment, integrate the information, and then coordinate a response appropriate to the

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environmental stimulus In addition to these basic vital functions, the nervous systems

of higher organisms are responsible for feeling, thinking, and learning All of the otherorgan systems of the body are subject to control by the nervous system; thus damage tothis “master” system by toxicants can have far-reaching and even devastating effects

In vertebrates there are two major components of the nervous system Althoughthese two systems are anatomically separable, they are contiguous and they functioninteractively The brain and spinal cord comprise the central nervous system (CNS),and the nervous tissue (ganglia and peripheral nerves) outside the brain and spinal cordcomprise the peripheral nervous system (PNS) The PNS can be further divided intothe somatic and autonomic nervous systems Somatic afferents carry sensory infor-mation from the skin, muscle, and joints to the CNS, while motor efferents innervateskeletal muscle to cause contractive movement The autonomic nervous system can bethought of as a motor system for visceral organs, since it projects to these organs toinnervate and control the function of smooth muscle, cardiac muscle, and endocrineand exocrine glands The autonomic nervous system is further divided anatomicallyand functionally into the sympathetic and parasympathetic subdivisions Most organsare innervated by both subdivisions, and their influences generally oppose one another.For example, stimulation of sympathetic nerves increases heart rate, while stimulation

of the vagus nerve, the primary parasympathetic innervation of the heart, slows its rate

of contraction

16.2.1 The Neuron

The basic unit of the nervous system is the neuron, a type of cell that is structurallyand functionally specialized to receive, integrate, conduct, and transmit information.Although neurons are far more diverse than any other cell type in the body, they dohave some common features Neurons are polarized cells; that is, they have differentcharacteristics on one end of the cell compared to the other (Figure 16.1) Typicallythe end of the neuron that receives information in the form of neurotransmitter stim-ulation from other neurons is highly branched into a region known as the dendritictree The branches are sometimes studded with projections, known as spines, whichcontain receptors that recognize and are activated by neurotransmitters It is here thatthe neuron is in close contact with other neurons via specialized structures calledsynapses Synapses are areas of close apposition where one neuron, the presynapticneuron, releases neurotransmitter into the gap, or cleft, between the two neurons Thepostsynaptic neuron then recognizes this chemical signal via receptors that are clus-tered in small densities opposing the presynaptic neuron Once the neurotransmittersignal is recognized by its receptors, the dendritic region of the neuron transmits theinformation as intracellular and electrochemical signals to the regions of the neuronwhere signal integration takes place In the typical neuron the arborizations of the den-dritic tree converge on the soma, or cell body, where the nucleus and most of RNA-and protein-synthesizing machinery exist The cell body usually then gives off a singleaxon, and it is in the region where the axon leaves the cell body (the axon hillock)that signals converge to be integrated into an all-or-none response

Neurotransmission down an axon is in the form of electrochemical signals In theresting state the interior of the axon is negatively charged with respect to the exte-rior The membrane is then said to be polarized, and the charge difference across themembrane in this resting state is approximately−70 mV Small depolarizing potentials

Figure 16.1 A neuron with accompanying astrocyte and myelinating oligodendrocyte.

arrive at the axon hillock from the dendritic regions where receptors have been ulated, and this stimulation results in the opening and closing of ion channels Thedepolarizing potentials occur primarily because of the opening of sodium channels,allowing sodium to transfer down its concentration gradient to the interior of the cell.Sodium brings with it a positive charge, and so the membrane in the region wherethe sodium channel opens becomes depolarized The depolarization is then detected

stim-by voltage-sensing sodium channels, which allow further influx of sodium When thespatial and temporal summation of these signals reaches a certain threshold (gener-ally about +50 mV), the axon will generate an action potential at the axon hillock.Once this occurs, all of the sodium channels in the vicinity open, allowing a mas-sive influx of sodium Sodium channels stay open for only a short period of time,and once they close, they cannot reopen for a while On the other hand, as sodiumchannels further down the axon sense the voltage change, they open, and thus a feed-forward effect is created The membrane is repolarized by the opening of potassiumchannels, which respond in a slightly delayed fashion, to the same signals that stim-ulate the sodium channels As the potassium channels open, potassium rushes out ofthe cell down its own concentration gradient This, combined with the closing of thesodium channels, produces a net efflux of positive charge, thereby repolarizing themembrane The process of depolarization/repolarization continues down the length ofthe axon In myelinated axons, the ion channels are clustered in regions between thesegments of myelin in regions known as nodes of Ranvier The myelin segments serve

to insulate the axon, and they allow the action potential to jump from one node to thenext, in a process called saltatory conduction (Figure 16.2) This results in much fasterpropagation of the action potential down the length of the axon

Axons terminate at neuromuscular junctions, in effector organs (e.g., the heart), and

in synapses with other neurons When the action potential reaches the terminal of theaxon, the depolarizing impulse stimulates the release of neurotransmitter from the ter-minal into the cleft between the presynaptic membrane and its effector or receiving

- +

-Na++ +

-

-K+

-Na++ + +

K+

- - - -

-

neuron The process of release usually involves the presence of packets of mitter called synaptic vesicles (Figure 16.3) These vesicles dock at the presynapticmembrane and, when stimulated to do so, fuse with the membrane to release their con-tents into the synaptic cleft The signal to fuse is thought to be primarily an influx ofcalcium, mediated by calcium channels on the presynaptic membrane that are sensitive

neurotrans-to changes in voltage Proteins on the vesicle membrane and the presynaptic membraneform complexes with one another, and when stimulated by the localized increase incalcium ion concentration, mediate the fusion of the two membranes and release ofneurotransmitter Electrical signals at work within the neuron are thus converted tochemical signals at work between neurons in the form of neurotransmitters

16.2.2 Neurotransmitters and their Receptors

Neurotransmitters are recognized by receiving neurons, neuromuscular junctions, orend effector organs via receptors that lie on the postsynaptic membrane Receptors aregenerally selective for the neurotransmitter that they bind The type of signaling that ischaracteristic of a given neurotransmitter is usually the result of the form of receptor towhich it binds For example, some receptors, like the nicotinic acetylcholine receptorfound in neuromuscular junctions, are ion channels The stimulation of the nicotinic

(a) (b)

Figure 16.3 Neurotransmitter release (a) Presynaptic nerve terminal containing vesicles and other organelles (b) Neurotransmitter-containing vesicles are made of lipid bilayers Associ- ated proteins participate in the release process (c) The vesicle associates with the presynaptic membrane via protein complexes that mediate release (d) Release of neurotransmitter into the

synapse is by protein-mediated fusion of vesicle and presynaptic membranes.

receptor by acetylcholine results in the opening of its channel, which is permeable

to sodium The influx of sodium then serves to depolarize the muscle membrane thatreceives acetylcholinergic innervation Neurotransmitter receptors that are ion channelsthus mediate very fast and short-lived neurotransmission, particularly when compared

to the other major type of neurotransmitter receptor, the G protein-coupled receptor

G protein-coupled neurotransmitter receptors activate intracellular signaling pathwaysthat produce a more slow and sustained response to neurotransmitter stimulation Ingeneral, these receptor-mediated pathways serve to modulate neurotransmission by ionchannels, maintain and mediate changes in protein expression, and promote cell sur-vival Most neurotransmitters have both ion channel receptors and G protein-coupledreceptors, although a few, like dopamine and norepinephrine, bind only to G protein-coupled receptors

Neurotransmitters stimulate receptors on postsynaptic membranes, but the signalsreceptors send are not always excitatory to the receiving neuron Receptors, directly orindirectly, modulate excitability of the postsynaptic neuron, so that it is more or lesslikely to fire an action potential For example, the neurotransmitter glutamate binds

to both ion channel receptors and G protein-coupled receptors, and in each case thesereceptors transmit a signal that enhances the excitability of the receiving neuron On the

other hand, the neurotransmitter GABA (for gamma-amino butyric acid), while also

binding to both types of receptors, is known for its ability to decrease the excitability

of the postsynaptic neuron Its message is therefore inhibitory to the propagation ofsignaling within a group of neurons

16.2.3 Glial Cells

While neurons constitute the definitive unit of the nervous system, their function iscritically dependent on the presence of glial cells In fact there are far more glial cells

in the nervous system than neurons Glial cells perform many functions, includingstructural support, insulation, buffering, and guidance of migration during develop-ment One class of glial cell, called microglia, is responsible for phagocytosis ofcellular debris following injury and infection The other types of glial cells, collec-tively known as macroglia, are the astrocytes and oligodendrocytes found in the CNS,and the Schwann cells found in the PNS Oligodendrocytes and Schwann cells formmyelin by wrapping multiple layers of plasma membrane around axons Astrocytes arethe most numerous of the glial cells, and they help form the blood-brain barrier, take upexcess neurotransmitter and ions, and probably have some nutritive function Metabolicenzymes expressed within astrocytes also regulate neuronal signaling by catabolizingexcessive amounts of neurotransmitter Monoamine oxidases, for example, catalyze thebiotransformation of dopamine, norepinephrine, and serotonin into oxidation productsthat are substrates for further enzymatic reactions en route to excretion The inciden-tal bioactivation of the xenobiotic MPTP to its neurotoxic metabolite MPP+ by theseenzymes will be discussed later in this chapter.

16.2.4 The Blood-Brain Barrier

The blood-brain barrier was conceptualized when it was noted that dyes injected intothe bloodstream of animals stained nearly all tissues except the brain It is thus thisbarrier and its PNS equivalent, the blood-nerve barrier, that prevents all but a selectfew molecules from entering the nervous system The barrier itself is not a singleunitary structure, but a combination of unique anatomical features that prevents thetranslocation of blood-borne agents from brain capillaries into the surrounding tissue

As mentioned above, astrocytes help form the barrier, surrounding capillary endothelialcells with extensions of their cytoplasm known as end-feet There are also pericytes,whose function is not well known, that associate with the capillaries and may helpinduce a functional barrier Another component of the barrier is the impermeablenature of the endothelial cells that line the interior of capillaries Capillary endothelialcells in the nervous system are different from those in the periphery in at least threeways First, brain capillaries form tight junctions of very high resistance between cells

In contrast, peripheral capillaries have low resistance tight junctions, and even ings, or fenestrations, that allow compounds to pass between cells Second, compared

open-to peripheral endothelial cells, brain endothelial cells are deficient in their ability open-totransport agents by endocytic mechanisms Instead, only small lipophilic particles can

be passed transcellularly For larger molecules, carrier-mediated transport mechanismsare highly selective, and allow only one-way transport Third, there is an enzymatic bar-rier that metabolizes nutrients and other compounds Enzymes such as gamma-glutamyltranspeptidase, alkaline phosphatase, and aromatic acid decarboxylase are more preva-lent in cerebral microvessels than in nonneuronal capillaries Most of these enzymesare present at the lumenal side of the endothelium, Additionally the P-glycoprotein(P-gp) drug efflux transporter is presently thought to exist at the lumenal membranesurface, although some scientists argue that P-gp is actually associated with astrocytes.Finally the CNS endothelial cell displays a net negative charge at its lumenal sideand at the basement membrane This provides an additional selective mechanism byimpeding passage of anionic molecules across the membrane

Most of the toxicants that enter the nervous system do so by exploiting anisms designed to allow entry of essential molecules, such as nutrients, ions, and

mech-neurotransmitter precursors Small, lipophilic molecules are able to cross the brain barrier relatively easily Some agents can be recognized by active transportsystems and thereby traverse the blood-brain barrier along with endogenous ligands.The Parkinson’s disease therapeutic agent levodopa enters the brain in this manner Insome cases the blood-brain barrier is itself subject to damage by neurotoxicants Forexample, lead, cadmium, mercury, and manganese accumulate in endothelial cells anddamage their membranes, leading to brain hemorrhage and edema.

blood-16.2.5 The Energy-Dependent Nervous System

Nervous tissue has a high demand for energy, yet nerve cells can only synthesize ATPthrough glucose metabolism in the presence of oxygen Critical ATP-dependent pro-cesses in the nervous system include regulation of ion gradients, release and uptake ofneurotransmitters, anterograde and retrograde axonal transport, active transport of nutri-ents across the blood-brain barrier, P-gp function, phosphorylation reactions, assembly

of mitochondria, and many others The highest demand for energy (up to 70%) iscreated by the maintenance of resting potential in the form of sodium and potassiumconcentration gradients across the nerve cell membrane These gradients are maintainedprimarily by the activity of the Na+/K+ ATPase pump The pump uses the energy ofhydrolyzing each ATP molecule to transport three sodium ions out of the cell, and twopotassium ions into the cell Maintenance of the resting potential is not the only benefit

of this pump’s activity, however The gradients created by the pump are also importantfor maintaining osmotic balance, and for the activity of indirect pumps that make use

of the sodium gradient to transport other molecules against their own concentrationgradient Neurotransmission is thus heavily dependent on the proper functioning of the

Na+/K+ ATPase pump

Another process dependent on energy metabolism is axonal transport Axonal port carries organelles, vesicles, viruses, and neurotrophins between the nerve nucleus

trans-ATP ADP

Figure 16.4 The microtubule motor protein kinesin Kinesin consists of two ATP-hydrolyzing subunits that contact microtubules, a stem region, and regions that interact with vesicle and organelle proteins One ATPase subunit binds and hydrolyses ATP, generating the force required

to advance it forward As this happens, the other subunit releases ADP, in preparation for binding another ATP, and its own advancement.

and the terminal This distance can be quite long when one considers that the length ofthe sciatic nerve, for example, can be up to one meter Anterograde transport (from cellbody to terminal) is accomplished by two mechanisms defined by their rate, fast axonaltransport and slow axonal transport Fast axonal transport proceeds at rates of approx-imately 400 mm/day, and is mediated by the ATP-dependent motor protein kinesin.Kinesin forms cross-bridges between vesicles or organelles and microtubules, and dualprojections of these cross-bridges shift back-to-front along microtubules in a coor-dinated, ATP-dependent manner, such that the entire molecule appears to be walking(Figure 16.4) Slow axonal transport is used to carry cytoskeletal elements such as tubu-lin and neurofilaments to the far ends of the axon, and it proceeds at approximately 0.2

to 5 mm/day Traditionally slow transport has been regarded as passively dependent onaxoplasmic flow; however, recent evidence suggests that the cytoskeletal elements actu-ally move rather quickly but frequently stall in a stop-and-go fashion Fast axonal trans-port also proceeds retrogradely, mediated by the ATP-dependent motor protein dynein.The rate of retrograde transport is about 200 mm/day Neurons use retrograde transportfor recycling membranes, vesicles, and their associated proteins Neurotrophic factors,and some viruses and toxins (e.g., tetanus toxin) are also transported by this mechanism

16.3 TOXICANT EFFECTS ON THE NERVOUS SYSTEM

Neurotoxicants affect the nervous system in a number of different ways Some toxicants damage the distal portions of axons without much effect on the remainder ofthe cell, while others produce outright cell death Still others affect signaling processes

neuro-in the nervous system, without causneuro-ing structural damage This wide variety of rotoxicant effects is due in part to the unique nature of the different types of neuronsand glia Neurons may be differentially vulnerable to certain neurotoxicants because

neu-of their functional characteristics, as in the case neu-of the targeting neu-of substantia nigraneurons by the active metabolite of MPTP, an agent that causes Parkinson’s disease.The substantia nigra, a brain region where neurons that synthesize dopamine are par-ticularly abundant, sends out axons that project to other parts of the brain where thedopamine is released After release, these neurons take back up the synaptic dopaminevia selective transporters on the nerve endings The damaging metabolite of MPTP,MPP+, is not distinguished from dopamine by the uptake transporter, so when present,MPP+ is taken up as well MPP+ kills substantia nigra neurons by affecting mito-chondrial energy production and promoting free radical formation The death of theseneurons results in a lack of dopamine release in an area of the brain called the stra-tum, which is responsible for the control of movement The loss of dopamine in thestratum causes the hallmark symptoms of the neurodegenerative disease Parkinsonism:slowed movement, rigidity, and tremors Because epidemiological studies have linkedParkinsonism in some patients with the agricultural use of pesticides, many of whichare toxic to mitochondria, scientists believe that at least some cases of Parkinson’sdisease may be related to long-term exposure to environmental toxins

MPTP is one example of a toxicant that causes direct structural damage to neurons,resulting in loss of function In the following sections, other types of structural andfunctional effects of neurotoxicants are described Structural effects are divided intothree primary types: effects on myelin formation, primary damage to axons, and directpromotion of cell death Neurons may also be secondarily affected by neurotoxicants

that target other cells in the nervous system, disrupting normal homeostatic function andcausing structural or functional damage Another method by which toxicants may affectthe function of the nervous system is by directly altering synaptic neurotransmission.

16.3.1 Structural Effects of Toxicants on Neurons

trans-duction Myelin acts like an electrical insulator by preventing loss of ion current, andintact myelin is critical for the fast saltatory nerve conduction discussed above Neu-rotoxicants that target the synthesis or integrity of PNS myelin may cause muscleweakness, poor coordination, and paralysis In the brain, white matter tracts that con-nect neurons within and between hemispheres may be destroyed, in a syndrome known

as toxic leukoencephalopathy A multifocal distribution of brain lesions is reflected inmental deterioration, vision loss, speech disturbances, ataxia (inability to coordinatemovements), and paralysis

Demyelination occurs secondary to axonal degeneration, a topic covered in thesection on axonopathy Neurotoxicants that produce primary demyelination are uncom-mon, but may be divided into those that affect the integrity of the myelin sheathwithout or prior to damage to the myelinating cells, and those that directly injuremyelin-producing cells The former include agents like hexachlorophene, isoniazid,and the organotins These compounds cause reversible edema between the layers ofmyelin by a mechanism that is yet unclear The optic nerve is particularly suscepti-ble to demyelination by hexachlorophene and organic solvents, whereas other cranialnerves, such as the trigeminal and vestibulocochlear, are vulnerable to styrene, xylene,and to trichloroethylene, an agent used in dry-cleaning The metalloid tellurium dam-ages myelin by inhibiting an enzyme involved in the synthesis of cholesterol, a majorcomponent of myelin

In contrast to agents that target the integrity of the myelin sheath, chronic sure to cyanide and carbon monoxide is thought to directly injure myelin-producingSchwann cell bodies in the PNS and oligodendrocytes in the CNS Inorganic lead alsocauses direct damage myelinating cells Oligodendrocytes appear more sensitive to leadtoxicity than astrocytes or neurons One mechanism for the devastating developmentaleffects of lead exposure may be the preferential inhibition of oligodendrocyte precursorcell differentiation

degen-eration of the axon, while leaving the cell body intact When axons die and degenerate,myelin breaks down as well, yet Schwann cells may survive and guide regeneration

of the axon in some cases Some toxicants produce axonal injury by directly targetingthe axon itself Others are thought to cause degenerative changes in axons by compro-mising the metabolic systems of the neuron In the latter case the distal portion of theaxon degenerates first, because it is this region that is most heavily dependent on intactaxonal transport mechanisms Since axonal transport is energy-dependent, toxicants thatinterfere with ATP production, such as the nicotinamide analogue Vacor, may causedistal regions to degenerate initially Agents that target tubulin, like the vinca alkaloids,also cause this type of injury, because the tubulin-derived microtubules are criticallyimportant for axonal transport With continued exposure the degeneration progresses

proximally, and eventually will affect the entire neuron This distal-to-proximal eration is called “dying back neuropathy,” and it affects the longest and largest diameterneurons most severely If exposure to the toxicant is discontinued before death of theentire proximal axon and cell body, axons in the PNS will often regenerate, but axonalregeneration does not occur within the CNS Regeneration in the PNS is dependent onSchwann cells proliferating and guiding growth of regenerating axon tips back to thetarget tissue.

degen-In the 1850s Augustus Waller described the sequence of events that occurred lowing transection of a nerve fiber These events have subsequently become known

fol-as Wallerian degeneration The essential features of this type of degeneration includeswelling of the axon at the distal end of the proximal segment of the transected axon,distal axonal dissolution and phagocytosis by inflammatory cells, and dissolution ofmyelin, with preservation and proliferation of Schwann cells along the length of theformer axon Certain neurotoxicants are capable of chemically transecting an axon,producing Wallerian degeneration similar to that occurring after slicing the nerve inhalf Hexane, for example, forms covalent adducts with neurofilament proteins Thischemical crosslinking is thought to result in neurofilamentous axonal swellings thatessentially block transport to regions of the axon distal to the swelling The distalregions then die due to lack of communication with the neuron cell body, undergoingWallerian degeneration

Axonopathy can manifest as defects in sensory or motor functions, or a combination

of the two For most neurotoxicants, sensory changes are noticed first, followed by gressive involvement of motor neurons One historically important case that illustratesthese effects is that of the epidemic poisoning resulting from the consumption of “Gin-ger Jake” during Prohibition Extracts of ginger used in tonics were legally required

pro-to contain 5 grams of ginger per milliliter of alcohol The Department of Agriculturechecked for compliance with this requirement by sampling the tonics, boiling off theethanol, and weighing the solid content Bootleggers soon discovered that a good deal

of money could be saved by cutting back on the ginger and substituting it with ating agents like castor oil and molasses It was such an attempt at adulterating GingerJake that led to the addition of Lyndol, a triorthocresyl phosphate (TOCP)-containingoil used in lacquers and varnishes, to tonic that was consumed by hundreds of thousands

adulter-of people The earliest signs that developed in people who had consumed the productwere noted days to weeks later, and began with tingling and numbness in the handsand feet In many, this progressed to leg cramps, weakness of the legs and arms, andataxia Those with minor symptoms improved, but perhaps thousands of people wereleft permanently paralyzed by the incident Today TOCP is used to study the syndrome

of delayed effects caused by some organophosphate compounds, commonly known asorganophosphate-induced delayed neuropathy (OPIDN) The nature of OPIDN is stillpoorly understood It appears not to be associated with organophosphate inhibition ofacetylcholinesterase, but rather with another neuronal enzyme, the neuropathy targetesterase (NTE) The physiological role of NTE is unknown

primary damage occurring at the nerve cell body Axonal and dendritic processes diesecondarily in response to loss of the cell body Like other cells in the body, neuronsdie by one of two processes distinguished by their morphological and molecular fea-tures: apoptosis and necrosis (This division is overly simplistic; there is much debate

over the characteristics of the two categories, and whether there are more than twocategories of cell death Nevertheless, only these two will be considered here.) Oftenthe same neurotoxicants can promote either form of cell death, depending on the inten-sity of the insult For example, methylmercury given to rats at a high dose for oneweek causes widespread histopathological damage consistent with necrosis, whereaslower doses spread out over a longer time period results in apoptotic changes restrictedprimarily to cerebellar granule cells It is thought that the severe and abrupt loss of cel-lular energy production by impairment of mitochondrial activity and plasma membranedisruption are responsible for necrotic death of neurons This affects surrounding tissuemore than apoptosis, since the dying cells release their contents and localized inflam-matory responses ensue On the other hand, apoptosis is death that is encoded withinindividual cells Apoptosis is characterized by a process of cell shrinkage, pyknosisand fragmentation of nuclei, and membrane budding The dying cell breaks apart intosmall membrane-bound apoptotic fragments that are phagocytosed, and thus collateraldamage is reduced because only cells with activated death programs are affected It

is important to remember that apoptotic and necrotic mechanisms of cell death canoccur concomitantly or sequentially, and thus are part of a continuum of effects asso-ciated with dose-dependent alterations in cellular energy production and the differentialsensitivity of neuronal subtypes

Many neurotoxicants produce their effects by promoting cell death in neurons.Neurotoxicant-induced cytotoxicity has been associated with the pathogenesis of anumber of neurodegenerative disorders, including Alzheimer’s disease, Parkinson’sdisease, and amyotrophic lateral sclerosis (ALS), and the weight of evidence sug-gests that toxicant exposure is a risk factor for these diseases One area of intenseresearch focus has been the toxic effects of excessive signaling by glutamate and otherexcitatory amino acids (EAAs), and the role that EAAs may play in neurodegener-ative disorders Glutamate activates ion channel receptors that open to allow influx

of calcium and other ions into the neuron This influx of ions, combined with othersecond messenger events that promote further intracellular release of calcium, con-tribute to calcium overload Signaling cascades are then activated in response to theintracellular calcium, and these pathways eventually lead to activation of oxidativestress and programmed cell death (apoptotic) pathways EAA-mediated neuronopathicinjury has been extensively studied for its role in ischemic and seizure-induced braindamage It is furthermore thought that the glutamate receptor agonist, domoic acid, atoxin produced by algae, is responsible for several outbreaks of shellfish poisoning

In one incident in 1987, several people died, and dozens became ill with dizziness,seizures, and memory loss after consuming blue mussels The mussels were contam-inated with domoic acid, found in high levels following an algae bloom near PrinceEdward Island, Canada More recently domoic acid produced by algae blooms hasbeen blamed for the abnormal behavior and deaths of pelicans, cormorants, and sealions on the California coast

16.3.2 Effects of Toxicants on Other Cells

Toxicants may selectively target glial cells for a number of reasons Myelinating glialcells constantly synthesize cholesterol and cerebroside for myelin production; thustoxicants that affect these synthetic pathways will preferentially affect myelination.The hydrophobic nature of myelin may serve as a reservoir for lipophilic toxicants

as well The specialized structures of cells that form myelin also present unique lenges to cellular homeostasis, increasing the vulnerability of these cells to toxicantaction.

chal-In general, a toxic or physical insult to either neurons or glial cells eventuallyleads to changes in the other cell type Because of this, it is often difficult to deter-mine whether the primary insult was neuronal or glial Metals such as lead, cadmium,and aluminum are capable of inducing cell death in cultured astrocytes and endothe-lial cells Methylmercury preferentially accumulates in astrocytes and to some extent

in microglia, causing cellular swelling The swelling is presumably the effect ofmethylmercury interfering with ion channels, because ion channel blockers can reversethis effect Astrocytes are important reservoirs of excess glutamate, and swollen astro-cytes release glutamate in and around synapses, potentially causing the excitotoxicitydescribed above Astrocyte swelling also has effects on brain blood flow, since astro-cyte end-feet surround the blood vessels of the CNS Not only does swelling result inreduced lumen size, but the distances substrates and waste products must diffuse toreach the bloodstream is increased

Astrocytes and microglial cells become activated secondarily to brain injury, such

as acute trauma or toxic lesioning This activation, known as “reactive gliosis” is acterized by astrocyte hypertrophy and hyperplasia Reactive astrocytes have greatlyenlarged cytoplasmic processes, and produce increased amounts of a protein known asglial fibrillary acidic protein (GFAP) GFAP is often used as a quantitative histochem-ical marker for toxicant-mediated injury in the nervous system

char-In addition to their function as phagocytic cells, glial cells produce neurotrophicfactors to prevent neuronal death and promote axonal growth after injury Glial cellsalso have xenobiotic biotransforming enzymes, but in some cases glial metabolismresults in xenobiotic activation to a toxic metabolite As discussed above, MPP+ isthe toxic metabolite of MPTP MPTP is taken up by astrocytes, where monoamineoxidase B converts MPTP to MPP+ While MPP+ seems to cause no damage tothe astrocytes themselves, the astrocytes release this reactive metabolite into synapses,where it is selectively taken up by dopamine re-uptake transporters on the endings ofneurons that normally release and recycle dopamine MPP+ then kills the dopamin-ergic neurons, and this insult to the movement-controlling brain circuitry results inthe classic motor symptoms of Parkinson’s disease The properties of MPTP haveonly become known since the 1980s, when its accidental ingestion by heroin addictsresulted in acute Parkinson-like symptoms These incidents spurred multiple investiga-tions, leading to much of what is known today about the pathogenesis of Parkinson’sdisease

16.3.3 Toxicant-Mediated Alterations in Synaptic Function

Nervous system function may be adversely affected by neurotoxicants without sarily causing structural damage to tissue In many cases neurotoxicants interfere withsignaling processes within the nervous system by activating or inhibiting receptors, oraltering the amount of neurotransmitter available to activate receptors This type ofneurotoxicity is illustrated by the well-characterized actions of the organophosphatesand carbamates on acetylcholine signaling

neces-Organophosphates inhibit acetylcholinesterase, the enzyme responsible for ing down acetylcholine into acetic acid and choline After acetylcholine has been

break-released into the synapse or the neuromuscular junction, acetylcholinesterase terminatesreceptor-stimulating activity by binding acetylcholine in its active site Separate siteswithin the binding pocket of acetylcholinesterase bind the quaternary nitrogen of thecholine group, and the carbonyl of the ester group A hydrolytic reaction results in theloss of choline, leaving an acylated serine residue, which is then rapidly hydrolyzed.The biologically active oxon forms of organophosphates also bind to the active site ofacetylcholinesterase, covalently phosphorylating the serine residue in the catalytic site

of the enzyme The phosphorylation of acetylcholinesterase creates a relatively stableinactive enzyme that persists for hours to days before hydrolysis of the phosphatemoiety occurs spontaneously, and acetylcholinesterase activity is restored The rate ofspontaneous hydrolysis is increased with larger alkyl groups attached to the phosphatemoiety When one or more of these alkyl groups is lost, in a process known as “aging,”spontaneous reactivation of acetylcholinesterase by hydrolysis of the phosphate moiety

is impossible, and the enzyme is permanently inactivated Carbamates similarly inhibitacetylcholinesterase by carbamylating the enzyme active site The stability of carbamy-lation is much less than phosphorylation, however, and spontaneous reactivation thusoccurs faster than with organophosphates

The effects of acetylcholinesterase inhibition can be seen throughout the nervoussystem Acetylcholine and its receptors mediate neurotransmission in sympathetic andparasympathetic autonomic ganglia, in the effector organs where autonomic nervesterminate, in neuromuscular junctions, and in the brain and spinal cord The signs ofhypercholinergic activity are thus very diverse, and include effects mediated by bothnicotinic and muscarinic types of acetylcholine receptor Hyperstimulation of nicotinicreceptors in neuromuscular junctions results in muscle weakness, in rapid, localizedcontractions called fasciculations, and in paralysis Nicotinic receptors are also found

in sympathetic and parasympathetic ganglia, and so stimulation of both divisions of theautonomic system is apparent as hypertension, increased heart rate, and papillary dila-tion Muscarinic receptors in the PNS mediate postganglionic parasympathetic effects

on the smooth muscle present in the end organs such as the lung, gastrointestinaltract, eye, bladder, and secretory glands Hyperstimulation of these receptors results

in a pattern of toxicity known by the mnemonic SLUDGE (salivation, lacrimation,urination, defecation, GI upset, emesis) Bronchospasm and bradycardia are also mus-carinic effects In the CNS, confusion, anxiety, restlessness, ataxia, seizures, and comaare effects of both muscarinic and nicotinic receptor overstimulation Death generallyoccurs from respiratory paralysis

Treatment for toxicity by organophosphates and carbamates is directed at teracting hyperstimulation and regenerating acetylcholinesterase enzymatic activity.Atropine is a muscarinic receptor antagonist (it blocks acetylcholine from binding tothe muscarinic receptor), and is used to counteract the effects of cholinergic overactiv-ity Atropine has no effect at the nicotinic receptor, however, so the skeletal muscularand some of the sympathetic effects of cholinergic hyperstimulation will remain afteradministration of atropine Inhibition of acetylcholinesterase activity by organophos-phates can be reversed by administration of oxime compounds (e.g., pralidoxime and2-PAM) These compounds contain a quaternary nitrogen that binds to the choline bind-ing site of acetylcholinesterase, positioning the oxime portion of the molecule near theesteratic site Oximes are themselves reversible inhibitors of acetylcholinesterase, buttheir mechanism of organophosphate reversal is by attack of the covalent phosphoser-ine bond, releasing the phosphate group Oximes are not effective on dealkylated or

coun-“aged” enzymes, so they must be administered soon after organophosphate intoxication

in order to be effective They are also ineffective against carbamate-mediated toxicity,and some researchers believe they actually worsen carbamate effects by stabilizing thecarbamylation of the enzyme

Many biological toxins produce hyperstimulation of receptors by directly ing and activating them (agonism), or reduce receptor stimulation by prohibiting theendogenous ligand from activating them (antagonism) A number of snake and spidervenoms, mushroom and plant alkaloids, affect nervous system function by these mech-anisms As the binding of receptors by these agents is usually reversible, their effectsare reversible as well (although some may still cause death by massively altering neu-ronal signaling) Beyond the receptor, an active area of current research is the role ofintracellular signaling molecules in mediating the effects of neurotoxicants The effects

bind-of a number bind-of metals, in particular, may be related to their ability to act as cbind-ofactorsfor proteins involved in intracellular signaling To date, however, few signal transduc-tion molecules have been shown to be directly affected by neurotoxicants Notableexceptions are cholera and pertussis toxins, which selectively target G proteins, buttheir primary effects are on the gastrointestinal and respiratory systems, respectively,rather than on the nervous system

On the other hand, the Clostridium toxins, botulinum (causing botulism) and spasmin (causing tetanus), block neurotransmission by inhibiting release of neuro-transmitter into synapses and at motor end-plates in muscle Both of these agents arestructurally similar proteases, but the effects they cause are vastly different Botulinumtoxin enters presynaptic motor neurons in the PNS, where it cleaves proteins that areinvolved in the fusion of synaptic vesicles with membranes This cleavage results inthe inhibition of acetylcholine release from the presynaptic terminal, and thus mus-cles cannot be stimulated to contract The clinical result of botulinum intoxication(usually by ingestion) is a flaccid paralysis Recovery occurs when the presynapticneuron sprouts new nerve endings that contact the muscle and create new motor end-plates Tetanus toxin causes a completely different clinical picture, even though itssubstrate specificity for cleavage of proteins is very similar Once taken up into thepresynaptic nerve endings, tetanus associates with endosomes, and like endosomes, istransported retrogradely toward the neuron cell body Tetanospasmin then continuesits trek to the dendritic regions of the neurons, where it is released, again retrogradely,into synapses Usually these synapses are within the spinal cord, where interneuronssend an inhibitory signal (via the neurotransmitters glycine and GABA) to motor neu-rons to slow their activity and prevent massive muscular contraction The presynapticmembranes of the interneurons take up tetanospasmin, and this is where most of itsactivity occurs By cleaving release-regulating proteins in the interneuron terminal,tetanospasmin prevents the release of glycine and GABA onto the motor neurons.The motor neurons then become hyperactive, and this results in overstimulation of themotor end-plate with acetylcholine Clinically this results in a spasms, stiffness, andwhole-body paralysis Again, the interneurons themselves do not die, but they mustform new synapses with the motor neurons Fortunately, in all but the most severecases, recovery is complete The reformation of new synapses by neurons, even in theCNS, is an example of the remarkable plasticity of the nervous system The continualformation and reformation of synaptic connections allows the organism to change andadapt to an inconstant environment

tetano-16.4 NEUROTOXICITY TESTING

A large number of the chemicals used in industry today remain poorly characterizedwith respect to their toxic effects on the nervous system In order to determine potentialrisks to human and environmental well-being, existing neurotoxicants must be identi-fied, and the approximately 2000 new chemicals introduced each year must be screenedfor their potential neurotoxic effects Often, a tiered approach is used, with the firsttier consisting of general screening tests to identify acute hazards The Environmen-tal Protection Agency (EPA) has proposed screening guidelines for tests in rodentsthat include a functional observational battery (FOB, see below) to evaluate sensory,motor, and autonomic effects, tests that identify changes in motor activity, and neu-ropathological assessment Interpretation of the outcome of tier 1 screening may lead

to more selective testing and examining the effects of repeated exposures in the secondtier Specialized tests for behavioral effects, developmental neurotoxicity, or delayedorganophosphate effects may be required If necessary, a third tier of testing char-acterizes mechanisms of neurotoxicant-induced injury Complete and comprehensiveevaluation of potential neurotoxicant effects requires that data from different types ofsources be considered; this can range from molecular interactions to whole animaland human exposure analysis Below are examples of techniques commonly used fortesting neurotoxic effects

16.4.1 In vivo Tests of Human Exposure

Historically the first indication of neurotoxic potential by a chemical has often followedaccidental human exposure in the workplace Case reports of incidents involving indi-viduals, or clusters of individuals, are useful for documentation but generally provide

a limited amount of information about the specific details of an exposure Proceduresincluded in most case reports include a patient medical history and clinical neuro-logical exam, sometimes supplemented with psychiatric or neurophysiological tests,and/or neuroimaging Although the specific tests involved vary depending on the clin-ician, most basic clinical neurological exams rely heavily on evaluation of mentalstatus (level of consciousness, orientation, mood, etc.) and sensorimotor function (gait,coordination, muscle tone, sensitivity to touch, reflexes)

Human epidemiological studies generally represent a deeper investigation into thecausal relationship between an exposure and neurotoxicological effects Some ofthe methods used to identify neurotoxic effects in epidemiological studies includebehavioral assessments, neurophysiological evaluations, and neuroimaging techniques.Neurobehavioral assessments examine a variety of psychological and cognitivefunctions such as mood, attention, memory, perceptual and visuospatial ability, andpsychomotor performance In an effort to standardize neurotoxicological testing ofhuman behavioral effects, particularly for studies involving worksite exposure, theWorld Health Organization (WHO) and the US National Institute for OccupationalSafety and Health (NIOSH) devised a the Neurobehavioral Core Test Battery (NCBT).The NCBT (Table 16.1) consists of seven tests that were shown previously to

be sensitive indicators of neurotoxicant exposure The battery is designed to beadministered one on one by an examiner Although this battery has a relatively narrow

Table 16.1 The WHO Neurobehavioral Core Test Battery (NBCT)

Psychomotor

performance

Motor speed, motor steadiness

Pursuit aiming Follow a pattern of small

circles, placing a dot

in each circle around a pattern; subject’s score

is number of taps in circle within one minute.

Manual dexterity, hand – eye coordination

Santa Ana Dexterity Test

Perform skillful movements with hands and arms.

Perceptual coding

and perceptual

motor speed

Wechsler Digit Symbol Test

Each number in a list is associated with a simple symbol On a list of random digits with blank spaces below them, write the correct symbols in blank spaces as fast as possible.

Attention and

short-term

memory

Attention and response speed

Simple reaction time

Test reactions of hands

or feet from visual and auditory signals Visual perception

and memory

Benton Visual Retention Test

Recall and reproduce figures.

Auditory memory Wechsler Digit

Span Test

Recall digits in series forwards and backwards immediately after hearing them.

States

Evaluate, by questionnaire, anger, tension, confusion, depression, etc.

focus, primarily on the effects most commonly seen in CNS toxicity, it also providessuggestions for the selection of further testing depending on the exposure setting TheNCBT has been widely used because of its ease of administration, relatively low cost,and its large base of control data A broader battery of cognitive and psychomotortests that is often used is the Neurobehavioral Evaluation System (NES) The NESconsists of a combination of automated (computerized) and hand-administered tests.The sensitivity of the NES to effects caused by neurotoxicants in industrial settingshas been validated internationally

Neurobehavioral examinations are useful for identifying neurotoxicant-mediateddeficits, but it is often difficult to localize the site of toxic action from such tests.For example, sensorimotor tests of reaction time, manual dexterity, hand-eye coordi-nation, and finger tapping can indicate either neuromuscular or psychomotor damage

The results of these tests thus should be interpreted in the context of other experiments.For example, electrophysiological techniques can help to focus an investigation to thesite of the lesion, and characterize electrical dysfunction within the damaged nerves.Electrophysiological nerve conduction studies can distinguish between proximal anddistal axonal lesions in peripheral nerves and can be performed noninvasively (i.e.,with skin surface electrodes) Characteristic changes in the velocity, duration, ampli-tude, waveform, or refractory period of peripheral nerves may be detected, depending

on the agent Evoked potentials represent another useful electrophysiological endpoint.These procedures measure the function of an entire system, such as the visual, audi-tory, or motor systems The specific pathway is stimulated by an evoking stimulus,such as a flash of light or electrical nerve stimulation The evoked potentials are read

as changes in ongoing electroencephalograms (EEGs) in response to the stimulation.Thus the activity of the entire neural circuit is evaluated in the brain after peripheralstimulation Evoked potentials can be very sensitive indicators of changes in neuralactivity when performed in a carefully controlled environment, and when interpreted

in light of behavioral or other physiological findings

An increasingly popular method of documenting brain pathology is the use of roimaging methods Computerized axial tomography (CAT) and magnetic resonanceimaging (MRI) can produce images of the brain that can show structural changes

neu-in the volume or density of a specific region or ventricle Other techniques, such aspositron emission tomography (PET) and single photon emission computerized tomog-raphy (SPECT), use radioactive tracer molecules to determine functional biochemicalchanges in processes like glucose utilization or receptor binding The number of cases

so far analyzed with neuroimaging techniques is still relatively small, and thus specifictoxicant-mediated effects are not well characterized Nevertheless, this growing fieldpromises to contribute significantly to neurotoxicity studies in the future

16.4.2 In vivo Tests of Animal Exposure

The primary approach currently used to detect and characterize potential neurotoxicantsinvolves the use of animal models, particularly rodents Behavioral and neurophysio-logical tests, often similar to the ones used in humans, are typically administered Thesensitivity of these measures to neurotoxicant exposure is widely accepted Although

it is often not possible to test toxicant effects on some higher behavioral functions inanimals (e.g., verbal ability, cognitive flexibility), there are other neurobehavioral out-comes such as memory loss, motivational defects, somatosensory deficits, and motordysfunction that can be successfully modeled in rodents These behaviors are based

on the ability of the nervous system to integrate multiple inputs and outputs, thus theycannot be modeled adequately in vitro Although the bulk of neurotoxicity data hasbeen collected in rodents, birds and primates are also used to model human behavioraloutcomes

As mentioned above, a useful screening tool for neurotoxicant exposure is a battery

of observational tests of function known as an FOB FOBs, like the one developed

by the EPA, are used to detect overt changes in behavior and physiology of animalsexposed to neurotoxicants In the typical exam, an observer documents cageside obser-vations regarding the appearance and activity of the animal Then the animal is handledand examined for obvious signs such as lacrimation, salivation, or piloerection Pupil-lary light responses and temperature are recorded, and the ease of handling the animal