Toxic Effects of Chemical Compounds

5.3.4.1 The Nature of Toxic Reactions

A toxic reaction may take place during or soon after exposure, or it. may only appear after a latency period. Chronic toxicity requires exposure of sev­eral years for a toxic effect to occur in humans. With respect to experimental animals, the animals are usually exposed for most or all of their life time to as­certain the occurrence of chronic toxicity. Acute toxic reactions that occur im­mediately are easy to associate with the exposure and the exposure-effect relationship can readily be demonstrated. The longer the time interval be­tween exposure and effect, the more difficult it is to delineate the relationship between exposure and effect.

Toxic effects often disappear after the cessation of the exposure, but they can also be permanent. The tissue’s ability to regenerate is one of the most im­portant factors that determines the nature of toxic effects. For example, liver tissue has a remarkable capacity to regenerate, and therefore liver injur)- is of­ten reversible. On the other hand, neuronal cells do not regenerate at all, thus neuronal injury is irreversible. It is true that neuronal cells can compensate for possible losses, but only to a minor degree. In particular, chronic effects tend to be irreversible.64

There are some basic differences between toxic and allergic reactions. The most important differences are: (1) an allergic reaction always requires a prior ex­posure to the compound, and this reaction only occurs in sensitized individuals; and (2) a dose-response relationship is characteristic to a toxic reaction, whereas such a relationship is much less clear for an allergic reaction. Even minute doses can elicit an allergic reaction in a sensitized individual (see Fig. 5.42).64

5.3.4.2 Antagonism and Synergism

Industrial workers are almost always exposed to several agents simulta­neously. The possible interactions of these multiple exposures are and will re­main (because the possible combinations are almost infinite) an area of great uncertainty. The situation is further complicated by the simultaneous presence of many lifestyle factors, especially smoking and the use of alcohol and drugs. Other exposures may enhance the toxic effect of an agent. The increased com­bined effect may be additive (1 + 1 =2) or synergistic (1 + 1 > 2). The neu­rotoxic effects of most organic solvents are usually considered to be additive; therefore, industrial bygiemsts use the combined exposure level to assess the conditions. It is obtained by dividing the concentration of each solvent by its OEL and by adding the quotients. If the sum exceeds one, the exposure is considered excessive. There are cases of synergism, where the toxic effects of individual exposures become greatly potentiated. A well known example is the combination of asbestos exposure and smoking. The various constituents may have no mutual interactions. In such a case, the effects of different agents can be considered individually. Since in most cases we are ignorant of these

Increase 111 infectivity/cancer Interference with t FIGURE 5.42 Responses of the immune system to exposure to some chemicals.65 (Used with permission.)

Potential interactions, the no-interaction assumption is the most common premise. Finally, it is also possible that some constituents reduce the effects of other exposures; however, there are no well-demonstrated examples of this kind of antagonistic action between occupational exposures. 5f>>57JS 5-87

The interactions may be physicochemical without the participation of bio­logical mechanisms; for example, deep lung exposure to highly soluble irrita­tive gases, such as sulfur dioxide, may become enhanced due to adsorption of the gas onto fine particles. Biological interactions may occur at all stages and body sites. For example, toxicity is increased when adverse effects are due to some reactive metabolic intermediate and exposure to another agent stimu­lates its metabolic activation (enzyme induction).

5.3.4.3 Mechanisms of Toxicity

Paracelsus, a Swiss physician of the sixteenth century, stated that every­thing is toxic, it is just the dose that matters.88 This statement still holds true 500 years after Paracelsus developed it to defend the use of toxic compounds such as lead and mercury in the treatment of serious diseases such as syphilis. Chemical compounds cause their toxic effects by inducing changes in cell physiology and biochemistry, and an understanding of cellular biology is a prerequisite if one wishes to understand the nature of toxic reactions.

Toxic reactions occur by several mechanisms: activation of metabolism, production of reactive intermediates and subsequent reactions with cell mac­romolecules, changing receptor responses, or through abnormal defence reac­tions. Several compounds cause toxicity by mimicking the organism’s own hormones or neurotransmitters, or activating the body’s endogenous receptors in some non-physiological way.89

Cells are capable of repairing minor damage, but extensive damage leads to cell death. This takes place through either necrosis, which is a chemical — driven chaotic and passive process, or programmed cell death, apoptosis, which is a genetically controlled and energy consuming process. Apoptosis is also a part of normal cell physiology in organogenesis during the development of the embryo before superfluous cells commit a form of cellular suicide by ac­tivating their apoptotic programs. However, many chemicals, e. g., several quinone oxidants, and heavy metals may overtly augment apoptosis in adults when it can turn into a pathological process. Thus, cell death is a crucial toxic injury which is affected by the rapidity of the cell injury as well as the target organ. It is noteworthy that the dose of a compound may determine whether cells will proliferate in a tissue or undergo apoptosis or necrosis (see Fig. 5.43 for necrosis and apoptosis).64’89’91

Cell response

NECROSIS

Differentiation Proliferation Quiescence Apoptosis

Internal information Genotype Cell type Metabolic state Cell damage Development history

External information Soluble signaling molecules Cell-cell interactions ^ Cell-substrate interactions Apogens

FIGURE 5.43 Left External and internal stimuli triggering various cellular responses including apto — sis. Right Comparison of morphologic characteristics of necrosis (toft) with apoptosis (bottom). A normal cell (top, A) usually begins the process of necrosis with an initial phase of generalized swelling (top, B), which progresses to a dissolution of organelles and rupture of plasma membranes (top, C). The earliest phase of apoptosis (bottom, A) involves retraction from adjacent cells, loss of specialized surface struc­tures, shrinkage with condensation of cytoplasm, margination of compacted nuclear chromatin, and localized protrusions of the cell surface. Nuclear fragmentation may occur at this time. In the next phase, the protuberances of the Cell surfaces separate into multiple membrane-bound bodies (apoptotic bodies) that contain nuclear remnants and intact organelles. The apoptotic bodies are then engulfed and degraded by resident tissue cells (bottom, C) or phagocytes. Note that the light microscopic appearances of nuclear rupture and chromatin disintegration (karyorrhexis) may be seen in both late necrosis (top, C) and apoptosis (bottom, 8). Modified from Searle et al.104

Cells in various tissues such as liver, kidney, or gastrointestinal tract, have a remarkable capacity to repair injuries inflicted by chemicals. Furthermore, the ability of most organs to fulfill their functions usually far exceeds require­ments that they need to perform. For example, humans can live with one lung, one kidney, and only part of their liver. In this regard, the central nervous sys­tem is an exception because neuronal cells do not regenerate. However, even neuronal cells are capable of compensating for an injury. This does nor take place through the replacement of dead cells but through the outgrowth of new extensions of existing neurons and through the formation of new synapses, i. e., contacts between neurons that allow chemical neurotransmission between neurons. Even though many toxic effects are due to cell death, toxicity may

Occur with functional consequences without there being any visible morpho­logical alterations in cells or tissues.89

Chemically induced changes in DNA, i. e., mutations and chromosomal damage, are also an important toxicity mechanism. The bases in DNA, like bases in general, are nucleophilic (electron donors) and react with electro — philes (electron acceptors). Strong electrophiles, such as carbonium ions and epoxides, are formed during the metabolism of many known potent carcino­gens. Thus, the formation of DNA adducts may cause malignant transforma­tion of cells and lead to initiation of cancer. In the following section, mechanisms whereby chemical compounds induce their toxicity will be dis­cussed.’7-89

Receptor-mediated Toxicity

Several chemical compounds induce their toxic and other effects through stimulating specific receptors and events occurring after receptor activation, a process called signal transduction. Receptors themselves are protein molecules sitting in the lipid bilayer of the cell membrane. They have the ability to recog­nize physiological intercellular transmitters such as hormones, neurotransmit­ters, or growth factors (also called first messengers). Normally a very small amount of a transmitter is sufficient to activate the receptor. Many of the re­ceptors are ion channels and their activation leads to the influx of ions into the cell. There are specific receptor-coupled receptors for sodium, potassium, and calcium. Increased influx of these ions usually leads to increased enzy­matic activity, and activation of the cell. Some receptors are intimately associ­ated with enzymes such as tyrosine kinase, adenylate cyclase, or phospholipase C.92-95

Some of the cell membrane receptors are coupled to an amplifier, called the G-protein. Activation of a G-protein leads either to activation or inhibi­tion of an effector enzyme on the internal side of the cell membrane (see Fig. 5.44).92 These effector enzymes are responsible for the generation of second messengers that are essential for cellular signal transduction. Whereas first messengers, described above, are responsible for chemical intercellular com­munication, second messengers are responsible for transducing the informa­tion that has reached the cell surface receptor to all parts of the cell interior. There is also a specific enzyme machinery for inactivating the second messen­gers to terminate the action that was initiated by the first messenger. Typical effects of a second messenger are elevation of free intracellular calcium associ­ated with cellular activation, activation of specific enzymes such as protein ki­nase ( . — or production of a tertiary cellular messenger such as nitric oxide (NO). Nitric oxide is a gaseous cellular messenger that can act as both an in­tra — and intercellular signal transduction factor. Being lipid-soluble, NO easily diffuses in the cell as well as penetrating through the cell membrane and thereby also reaching other cells.96 97

There are even receptors that are known to become activated only due to interaction with a synthetic chemical, and no physiological agonist for such a receptor has been characterized. A model receptor in this class is the so-called Ah receptor complex that becomes activated subsequent to its exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Activation of the Ah receptor

Ins IP

L^V

CDP. DAG

Inositol

Ca* V’,pf (m:(|MP0 RЈ^Dr VWM’ > Cell Membrane U, v 1 *"M s (c **) Calcium stores

Iо lns4P I

FIGURE 5.44 Acetylcholine (A) binds to a receptor (R) coupled to a G-protein (Gp), and stimu­lates phospholipase C (PLC). PLC hydrolyzes phosphatidylinositol-(4,5)-b is phosphate (PIP2) to inositol — (l,4,5)-triphosphate (lns(I.■4.5)P3) and diacylglycerol (DAG). DAG stimulates protein kinase C (PKC), and lns(l,4,5)Pj binds to its receptor (R) in the intracellular Ca2* store, releases Ca2’. and elevates the levels of free intracellular calcium ([Ca2*]). lnositol-(l,3,4,S)-tecracisphosphatc (lns(l.3,4,S)P<) is formed from lns(M,5)Pj by phosphorylation, and it. together with Ins(H. S)P) controls influx of Ca2* PKC and Ca!* cause neuronal stimulation.52 (Used with permission.)

Complex by TCDD leads to increased expression of multiple genes that encode for many proteins such as the enzymes belonging to the phase I CYP P450 family (see Fig. 5.45). This may cause major alterations in the biotransforma­tion of xenobiotics.89’98-100

In the CNS, acetylcholine (ACh) is one of the key excitatory neurotrans — mitrers (first messengers). In neuronal cells, it binds preferentially to muscar­inic receptors. Stimulation of muscarinic receptors increases levels of neuronal free intracellular calcium leading to neuronal activation. If present in excess, ACh leads to epileptiformic seizures and tonic-clonic convulsions which may be associated with neuronal injury.92’101,102 Glutamate (Glu) is the most ubiq­uitous excitatory neurotransmitter in the CNS which binds to several subtypes of glutamatergic receptors. Glutamate is released from nerve endings of glutamatergic neurons subsequent to neuronal stimulation, and also during hypoxia and neuronal injury. Glu also elevates neuronal levels of free intracel­lular calcium, activates cells, and, when in excess, can cause neuronal injury.9i Thus, both of these molecules are endogenous neurotransmitters which in ex­cess are harmful to the CNS. Occupational and environmental contaminants such as lead may amplify the effects of Glu, and thereby cause severe neuro­toxic risks to exposed individuals.103,104

One of the important consequences of neuronal stimulation is increased neuronal aerobic metabolism which produces reactive oxygen species (ROS). ROS can oxidize several biomolecules (carbohydrates, DNA, lipids, and pro­teins). Thus, even oxygen, which is essential for aerobic life, may be poten­tially toxic to cells. Addition of one electron to molecular oxygen (02) generates a free radical (O^), the superoxide anion. This is converted through activation of an enzyme, superoxide dismutase, to hydrogen peroxide (H202), which is, in turn, the source of the hydroxyl radical (OH). Usually catalase

Further metabolizes hydrogen peroxide to molecular oxygen and water. Oxy­gen may also be activated to the highly reactive singlet oxygen. It is important to note that the formation of reactive oxygen species is a part of normal cell respiration, i. e., in electron transfer during metabolism of oxygen by the CYP enzyme system. During this process, a part of the ROS formed leaks into the

Production of ROS is not only a detrimental process; several cells carry out their functions in the body by generating ROS. For example, neutro­phils and macrophages produce ROS upon activation. This is one of their ways of destroying invading microorganisms. However, other exposures, such as mineral fibers, and inorganic and biological particles are also able to activate phagocytes to produce ROS. Excessive ROS production may be harmful to the host cell and surrounding ceils.9’’101

Cells have defense systems to protect themselves against these radical spe­cies. The defense systems constitute intracellular thiols, such as glutathione (GSH), a molecule rich in SH groups, and thus capable of scavenging the reac­tive species through oxidation of the SH groups. This oxidation leads to the formation of disulfide bridges; oxidized GSH (GSSG) is unable to scavenge oxygen radicals. GSH has to be regenerated and this reduction is performed by a specific enzyme, glutathione reductase.105 Cells also contain water — and lipid soluble molecules that remove ROS. The most important ot these mole­cules are a water-soluble vitamin, vitamin C, that acts in the cytoplasm, and a lipid-soluble vitamin, vitamin E, that functions in the cell membrane. (leiluLir defense mechanisms against excessive production of ROS also include en­zymes that metabolize these reactive species; superoxide dismutase (SOD) me­tabolizes superoxide anion to hydrogen peroxide, and catalase breaks down hydrogen peroxide to molecular oxygen and water. Oxidative stress results when activation of cells leads to such a high production of ROS that it over­whelms the capacity of the defense mechanisms. The initial phases of stress are associated with depletion of cellular GSH. Then the depletion of defense vitamins C and E occurs. This means that vital biological macromolecules, no­tably DNA, proteins, carbohydrates, and lipids, can be attacked by the reac­tive species. This cascade of events may lead to cell death through necrosis or apoptosis.104-106

Effects on Excitable Membranes

Maintenance of electrical potential between the cell membrane exterior and interior is a necessity for the proper functioning of excitable neuronal and muscle cells. Chemical compounds can disturb ion fluxes that are essential for the main­tenance of the membrane potentials. Fluxes of ions into the cells or out of the cells can be blocked by ion channel blockers (for example, some marine tox­ins).107

Insecticides, such as DDT and lindane, cause their neurotoxic effects by affecting the functions of ion channels in the neuronal cell membrane, thereby altering depolarization of the cell membrane. Organic solvents also modify the normal functioning of excitable neuronal membranes. It was originally as­sumed that organic solvents non-specifically altered the fluidity of the cell membrane. Current knowledge is that the effects of organic solvents are more specifically directed toward cell membrane proteins such as ion channels, other receptors, and specific enzymes.107

Effects on Cellular Energy Metabolism

Several toxic compounds act by inhibiting the oxidation of carbohydrates or by inhibiting the formation of adenosine triphosphate (ATP), a molecule that

Is an essential energy source of the cells. Cellular energy metabolism can be pre ­vented by inducing anoxia, for example, by exposure to carbon monoxide. 89 Carbon monoxide reacts with hemoglobin and forms carboxyhemoglobin, which is unable to bind oxygen. Nitrite-induced oxidation of heme iron causes formation of methemoglobin from hemoglobin. An increased amount of methe- moglobin also prevents oxidation of cells and tissues because methemoglobin does not bind oxygen. However, the treatment of methemoglobinemia is much easier than the treatment of CO-induced carboxyhemoglobinemia. In fact, in­duction of slight methemoglobinemia with nitrite can be used as an antidote in cyanide poisonings, because the ferric (trivalent iron) form present in methemo­globin acts as a sink by binding free cyanide.108

Cyanide, hydrogen sulfide, and azides prevent cells and tissues from utiliz­ing oxygen by binding to cytochrome oxidase and thereby preventing mito­chondrial energy production. The release of hydrogen cyanide may take place if cyanides make contact with acids, and, for example, sewage workers may be exposed to hydrogen sulfide if anaerobic conditions occur. Formation of hy­drogen sulfide also takes place in many industries. This gas is insidious be­cause its very unpleasant odor virtually disappears at high concentrations.109 The inhibition of ATP formation can also take place through other mecha­nisms. Dinitrophenol (a herbicide) blocks the citric acid cycle by uncoupling it from mitochondrial oxidative metabolism. Fluoro-aceticacid, in turn, blocks the citric acid cycle by inhibiting several key enzymes in the cycle.99’110’111

Depletion of ATP in the cells prevents maintenance of the membrane po­tential, inhibits the functioning of ion pumps, and attenuates cellular signal transduction (e. g., formation of second messengers such as inositol phos­phates or cyclic AMP). A marked ATP depletion ultimately impairs the activ­ity of the cell and leads to cell death.

Disturbances in Cellular Calcium Metabolism

As stated above, calcium is an extremely important cellular ion for several cellular functions. The concentration of calcium in human extracellular fluid is about 2.5 mM, while the intracellular concentration is only 100-200 nM depending on the cell type. Thus, there is 10 000-20 000 fold concentration difference between the cell interior and exterior that has to be maintained by cellular pumping mechanisms. This requires a large amount of energy.93-112 51 !

The behavior of calcium in the cells can be considered as a metabolic pro­cess. There is uptake, distribution, and excretion of calcium in the cells. The uptake of calcium occurs via activation of calcium channels. The end result is elevation of intracellular calcium levels and subsequent activation.89-91-*14 Be­cause calcium is a powerful cell-activating ion, increased calcium levels in the cell have to be controlled carefully. There are a number of calcium pumps that are responsible for pumping of calcium out of the cells. This again requires a large amount of energy.1,3

Nitric Oxide

Nitric oxide (NO) is a gaseous cellular messenger that transmits informa­tion between cells and within cells. In spite of its physiological role, NO is also a reactive species which is capable of reacting with biological molecules, and therefore in some instances tissue damage may ensue. NO is produced by an en­zyme, nitric oxide synthase (NOS) which acts on arginine, transforming it into citrulline and NO. This enzyme has both inducible and constitutive forms. In­ducible NOS {iNOS) is expressed in immunological cells, mainly phagocytes such as macrophages and neutrophiles, and in epithelial cells of the airways as well as endothelial cells of the circulatory system.97’116 Constitutive NOS (cNOS) is expressed in many cells e. g. neuronal cells. It is characteristic of iNOS that NO is produced only subsequent to persistent induction of the enzyme. Upon stimulation, the induction of iNOS (stimulated Synthesis of the enzyme protein) may take several hours, but after this time period, the cell can produce large amounts of NO. When airway epithelial cells and circulatory endothelial cells produce NO, they contribute to the control of the tone of the smooth mus­cle in these systems and thus modify airway resistance and blood pressure.97 NO production is associated with asthma and airway infections; in both situa­tions, an increased concentration of NO can be measured in the exhaled air.96

On the other hand, cNOS is continuously expressed in the cells, and upon stimulation of the cell, the formation of NO begins immediately. However, the amounts of NO produced are minute. The nature of NO in cells expressing cNOS is only to act as a messenger molecule, whereas NO has also other func­tions in cells expressing iNOS. For example, NO has bacteria and cell killing properties in immunological cells, such as phagocytes.97,116,117

Nitric oxide may induce deleterious effects when airway epithelial or im­munological cells are exposed to mineral particles (asbestos, quartz). These particles also stimulate cells to produce NO in large quantities, but pulmonary cells are unable to destroy these particles, and a non-physiologically excess production of NO results, perhaps causing tissue damage due to a reaction of NO with cellular macromolecules.118119

Immunological Responses and Sensitization

A number of chemical compounds are potent sensitizers that can lead to seri­ous immunological reactions. Immunotoxicology explores interactions between chemical compounds and the immune system. Chemicals can amplify, attenuate, or otherwise modify immunological reactions subsequent to exposure.120

The basic function of the immunological system is to detect and destroy foreign material that may be harmful to the organism. Cells that belong to the immunological system include macrophages, monocytes, granulocytes, and T­and B-lymphocytes. All cells that belong to the immune system have differenti­ated from the same stem cell. In harmful immunological reactions, the response of an organism to an exposure changes. The environmental factor does not act directly, but alters the reaction of the person exposed. The most important forms of this kind of immunological reactions are (1) immunosuppression; (2) uncontrolled cell growth, e. g., leukemia and lymphoma; (3) disturbances of im­munological defense mechanisms against infectious agents and malignant cells; (4) allergies; and (5) autoimmunity. Allergies will be dealt with ш more detail later in this chapter.120

Necrotic and Apoptotic Cell Death

The main types of cellular injury induced by chemical compounds are ne­crotic and apoptotic (programmed) cell death. Necrosis implies chaotic ending

Of cellular functions, and it always represents an unwanted effect on the cell by a chemical. Apoptosis is a physiological phenomenon that is required dur­ing development of the embryo in shaping the developing organs into their fi­nal size and form, and it is also functionally important in the development of organs and even body parts (e. g., fingers and toes). Apoptosis is also impor­tant in maintaining the integrity and renewal of mucous membranes and the skin. In direct contrast to necrosis which is a passive, non-energy-requiring phenonomenon, apoptosis requires gene expression and synthesis of new pro­teins, and it is an energy-expensive process.89’91

Necrotic cell death is often due to binding of reactive species to biologi­cally important cellular macromolecules, such as proteins, lipids, and DNA. Biotransformation of a number of chemicals such as carbon tetrachloride or styrene leads to formation of epoxides that bind to nucleophilic sites on pro­teins and DNA. Many of these compounds are also carcinogens. Furthermore, several compounds also cause increased production of ROS. These phenom­ena may also damage the cell membrane, leading to its leakage and rupture. Necrosis is characterized by cell swelling and leakage of cell constituents into the surroundings of the cell.

In apoptotic cell death, several factors such as growth factors, NO, the tu­mor suppressor gene p53, and the protein encoded by this gene contribute to the process that leads to cell death. One of the functions of p53 protein is the activation of apoptosis if a cell is transformed to a malignant cell. Apoptosis typically leads to the formation of smaller membrane-encapsulated particles within the cell. Apoptotic cell death begins in the nucleus and proceeds to other parts of the cell. The death process may be quite advanced before it can

TABLE 5.10 Some Important Biochemical Events in Apoptotic Control91

1. The detachment of chromatin from the nuclear scaffold, leading to chromatin

Condensation.

2. Endonuclease-catalyzed hydrolysis of DNA at the internucleosomal linker

Regions into multimers of 180 base pairs which are visualized by electrophoresis as a “ladder” of nuclear DNA fragments. Access of the endonuclease to DNA is facilitated by depletion of polyamines, and the activity of the enzyme is increased by Ca2+ and decreased by ADP-tibosylation. Thus, agents that increase intracellular Ca2+ or inhibit poly(ADP-ribose) polymerase can induce apoptosis.12’•

3. Induction of transglutaminase, an enzyme that cross-links proteins through

Ј-(7-glutamyl)lysine bonds and presumably contributes to the formation of membrane-bound apoptosis bodies.

4. Protein kinase A activation usually promotes, whereas protein kinase C

Activation retards, apoptosis.

5. Increased synthesis of transforming growth factor-beta 1, which blocks cell

Division and promotes apoptosis by interacting with its own membrane receptor.122

6. Cytotoxic T lymphocytes induce apoptosis of target cells by producing the

Fas ligand, a signaling protein that activates Fas, a membrane receptor on potential target cells, including those of the liver, the heart, and the lungs.123

Be observed from outside the cell. Ultimately, the cellular particles are phago — cytized by the surrounding cells without any inflammatory process. This is one of the characteristic morphological differences between necrotic and apo — ptotic cell death: whereas inflammation is typical for necrosis, lack >>f inflam­mation is the hallmark of apoptosis.89,91 See Table 5.10 for features of apoptosis. Table 5.11 lists events important for necrosis.

Exposure to chemical compounds such as some heavy metals (e. g.> lead) may activate apoptosis in a non-physiological way, leading to organ injury

TABLE 5.11 Agents Causing Sustained Elevation of Cytosolic Ca2+ and/or Impaired Synthesis of ATP

A. Agents inducing Ca2* influx into the cytoplasm

I. Via ligand-gated channels in neurons:

1. Glutamate receptor agonists (“excitotoxins”): glutamate, kainate,

Domoate

2. “Capsaicin receptor” agonists: capsaicin, resmiferatoxin II. Via voltage-gated channels: maitotoxin (?) OH*

III. Via “newly formed pores”: maitotoxin, amphotericin B, chlordecone,

Methylmercury alkyltins

IV. Across disrupted cell membrane:

1. Detergents: exogenous detergents, lysophospholipids, free fatty

Acids

2. Hydrolytic enzymes: phospholipases in snake venoms, endogenous

Phospholipase A,

3. Lipid peroxidants: carbon tetrachloride

4. Cytoskeletal toxins (by inducing membrane blebbing):

Cytochalasins, phalloidin

V. From mitochondria: see D

B. Agents inhibiting Ca2+ export from the cytoplasm (inhibitors of Ca2+-ATPase in

Cell membrane and/or endoplasmic reticulum)

I. Covalent binders: acetaminophen, bromobenzene, CC14,

Chloroform, DCE

II. Thiol oxidants: cystamine (mixed disulfide formation), diamide, /-BHP,

Menadione, diquat

III. Others: vanadate

C. Agents impairing mitochondrial ATP synthesis

I). Agents causing hydrolysis of NAD(P)+ in mitochondria

I. By increasing NAD(P)* availability via oxidation of NAD(P)H

1. Directly: alloxan

2. Enzymatically: Ј-BHP, NAPBQI, divicine, fatty acid hydroperoxides,

Menadione, MPP+

II. By activation of “NAD-glycohydrolase”: phenylarsine oxide, gliotoxin, NO*

DCE = 1,1-dichloroethylene; Ј-BHP = /-butyl hydroperoxide;

MPP* = 1 -methyl-4-phenylpyridinium; NAPBQI = N-acetyl-p-benzoquinoneimine. Source: Modified from Gregus and Klaassen.*9

And reduced functional capacity of the organ. It is noteworthy that effects of various oxidants, such as quinones, can vary as a function of dose: at low doses they may induce cellular proliferation, at moderate doses apoptosis, and at high doses they induce necrosis. Thus again dose is the ultimate determi­nant of the effect, even when very basic cellular responses such as death or survival are involved.124

Binding to Cellular Macromolecules

Many chemical compounds induce their toxic effect by binding to the ac­tive site of an enzyme or to other proteins that are vital for cellular functions. As described above, hydrogen sulfide and cyanide bind to the Fe3+ of cyto­chrome oxidase whereas carbon monoxide binds to the Fe2+ of hemoglobin.89 Consequently, cyanide prevents a cell from utilizing oxygen even if it would be available and carboxyhemoglobin formation during CO exposure inhibits the access of cells to oxygen and thereby terminates oxidative metabolism inside the cells. Lead, mercury, and cadmium bind to SH-groups of proteins and thereby inhibit their functions.89’125 A classic example of fatal enzyme inhibi­tion is the covalent binding of organophosphate insecticides, such as the acti­vated form of parathion, paraoxon, to the acetycholinesterase enzyme. This leads to accumulation of acetylcholine in the central nervous system, endo­crine glands, smooth muscle, and other organs. This, in turn, leads to clinical signs such as breathing difficulties, excessive salivation, tremors, convulsions, and even death.126’127 The mechanism of this enzyme inhibition is illustrated in Fig. 5.46.

Covalent binding of chemicals to biological macromolecules can also cause toxicity. During biotransformation and metabolic activation, chemi­cal compounds can be changed to free radicals, which have an unpaired

Organophosphate

TOC o "1-5" h z xo O XO XO o

^ ^

OH + P —► E — OH — P — O —► Ј — OH + P ‘

/ / /

YO Z YO YO OH

+ ZH

Carbamate

O O o

Il II II

K —OH + XOC — NHCH^—► L— O — C— NHCH, —► E — OH + HO—C — NHCH„

+ XOH ‘

FIGURE 5.46 Interaction of the serine hydroxyl residue in the catalytically active site of acetylcho­linesterase enzyme with esters of organophosphates or carbamates. The interaction leads to binding of the chemical with the enzyme, inhibition of the enzyme, inhibition of acetylcholine hydrolysis, and thus accumulation of acetylcholine in the synapses.

Electron. These are extremely reactive, and readily react with cellular lipids, causing lipid peroxidation, where polyunsaturated fatty acids are converted to lipid peroxyradicals that are further changed to lipid hydroxy-peroxides. These are then the source for lipid peroxides. This is a typical chain reac­tion that continues until it is stopped by antioxidants.128 If there is j short­age of antioxidants in the cell e. g., due to oxidative stress that has depleted GSH, the end result may be cell death. Thus, intracellular thiols, especially glutathione, are extremely important in preventing radical-induced cellular injuries.128 Figure 5.47 depicts the role of glutathione in the protection of cells against attack by electrophiles, oxidants, and reactive oxygen spe­cies.64

Nucleic acids in the DNA contain a high number of nucleophilic sites that can be attacked by electrophilic intermediates (metabolites) of chemical com­pounds. DNA adducts formed may cause alterations in the expression of a critical gene in the cell and thus lead to cell death. For example, modification of p53 tumor suppressor gene may inactivate the functions of the p53 protein and render cells sensitive to malignant transformation. Also, formation of RNA adducts may inhibit key cellular events because RNA is essential for pro­tein synthesis.

Glutathione reductase

Biosynthesis of GSH

GSH GSH conjugate

Protein nucleophiles f (thiol, histidine, lysine) r__JU5k.

Modified protein Intracellular

Protein biosynthesis 7

To extracellular

Kidney (mercapturic acid for excretion)

Biliary

Excretion

FIGURE 5.47 The role of glutathione and metabolic pathways involved in the protection of tissues against intoxication by electrophiles, oxidants and active oxygen species.65 (Used with permission.)

Genetic Damage

A genetic injury often leads to the formation of inactive protein or inhibi­tion of synthesis of a certain protein. There are endless possibilities for such interactions between DNA and chemical compounds because each human cel! contains about 100 000 genes. Genetic damage leads to an inheritable injury only when it occurs in a germ cell that is involved in fertilization and develop­ment of a new organism. Genetic damage in a somatic cell may lead to a dele­terious effect in an individual since it can ultimately lead to a toxic end result such as cancer.

Most of the compounds that induce alterations in genetic material, i. e., mutagens, also induce cancer, i. e., they are also carcinogens. For this reason, mutagenicity tests have been widely used to predict carcinogenicity. They are also used for biological monitoring of exposed workers to identify early dam­age to the genetic material in human cells. It has to be noted, however, that re­cently mutagenic tests have been heavily criticized for a number of reasons, and a positive result in a single mutagenic test can never be considered as a clear indication of carcinogenicity or even mutagenicity. Instead, a combina­tion of several mutagenicity tests all producing positive results is clearly a cause for concern.129

Genetic damage can take place at the level of the chromosome or at the gene level. In addition, chemicals can also induce alterations in the number of chromosomes in the cells. Aneuploidy is an excess or a shortage of a single chromosome. Polyploidia is an excess of a whole set of chromosomes in the cell.

Chromosomal aberrations represent damage to the chromosomal struc­ture that can be detected microscopically. The most frequent chromosomal ab­errations are deletion (lack of a chromosome or its part), duplication (part of a chromosome has been duplicated), inversion (parts of a chromosome have changed place within that particular chromosome), and translocation (parts of chromosomes have changed their position between two chromosomes). Many of these chromosomal changes are transferred to sister cells when the cell di­vides, and become, therefore, stable chromosomal aberrations. Cytostatic drugs and cigarette smoke are examples of chemical exposures known to in­duce chromosomal aberrations. Chromosomal aberrations themselves do not, however, give any clue of the causative agents for the changes.129

Genotoxic compounds can induce a number of different mutations. A gene or a part of a gene can be missing (deletion ), additional genetic material may become added to a gene (insertion), a gene may be amplified (amplifica­tion), or a genetic change may concern only one nucleotide, a basic structural unit of nucleic acid. The latter change causes deletion of one single amino acid in a protein encoded by the gene, and may lead to inactivation of a pro­tein. The result is a frame-shift mutation if a number of nucleotides (usually one or two) upset the regular arrangement of the three nucleotide-code. This kind of change alters the amino acids throughout the protein because each amino acid has its own code consisting of three nucleotides. A number of chemical compounds bind to DNA, and may cause point mutations. Ionizing radiation-induced DNA damage typically causes deletions. Table 5.12 lists the principal assays used in genetic toxicology.130

I. Pivotal assays

A. An assay tor gene mutations

SdmomllaltMimmaXian microsome assay (Ames test)

B. A mammalian assay for chromosome damage in vivo

Metaphase analysis or micronucleus assay in rodent bone marrow

II. Other assays offering an extensive database or unique endpoint

A. Assays for gene mutations

Ј. colt WP2 trytopban reversion assay

TK or HPRT forward mutation assays in cultured mammalian ceiis Drosophila sex-linked recessive lethal assay

B. Cytogenetic analysis in cultured Chinese hamster or human cclis

Assays for chromosome aberrations and micronuclei Assays for aneuploidy

C. Other indicators of genetic damage

Assay for mitotic recombinarion in yeast and Drosophila Assay for unscheduled DNA synthesis in cultured hepatocytes and rodents

D. Mammalian germ cell assays

Mouse visible or electrophoretic specific-locus tests Assays for skeletal and cataract mutations Cytogenetic analysis and heritable translocation assays DNA damage and repair in rodent germ cells Dominant lethal assay

Source: Modified from Hoffmann.110

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