Chapter 3. Mechanisms of Toxicity

• Toxicity involves toxicant delivery to its target or targets and interactions with endogenous target molecules that may trigger perturbations in cell function and/or structure or that may initiate repair mechanisms at the molecular, cellular, and/or tissue levels.
• Biotransformation to harmful products is called toxication or metabolic activation.
• Biotransformations that eliminate the ultimate toxicant or prevent its formation are called detoxications.
• Apoptosis, or programmed cell death, is a tightly controlled, organized process whereby individual cells break into small fragments that are phagocytosed by adjacent cells or macrophages without producing an inflammatory response.
• Sustained elevation of intracellular Ca2+ is harmful because it can result in (1) depletion of energy reserves by inhibiting the ATPase used in oxidative phosphorylation, (2) dysfunction of microfilaments, (3) activation of hydrolytic enzymes, and (4) generation of reactive oxygen and nitrogen species (ROS and RNS).
• Cell injury progresses toward cell necrosis (death) if molecular repair mechanisms are inefficient or the molecular damage is not readily reversible.
• Chemical carcinogenesis involves insufficient function of various repair mechanisms, including (1) failure of DNA repair, (2) failure of apoptosis (programmed cell death), and (3) failure to terminate cell proliferation.

An understanding of the mechanisms of toxicity provides a rational basis for interpreting descriptive toxicity data. The cellular mechanisms that contribute to the manifestation of toxicities are overviewed by relating a series of events that begins with exposure, involves a multitude of interactions between the invading toxicant and the organism, and culminates in a toxic effect.

As a result of the huge number of potential toxicants and the multitude of biological structures and processes that can be impaired, there are a tremendous number of possible pathways that may lead to toxicity (Figure 3–1). Commonly, a toxicant is delivered to its target, reacts with it, and the resultant cellular dysfunction manifests itself in toxicity. Sometimes a xenobiotic does not react with a specific target molecule but rather adversely influences the biological environment, causing molecular, organellar, cellular, or organ dysfunction leading to deleterious effects.

The most complex path to toxicity involves more steps (Figure 3–1). First, the toxicant is delivered to its target or targets (step 1), interacting with endogenous target molecules (step 2a) or altering the environment (step 2b), triggering perturbations in cell function and/or structure (step 3), which initiate repair mechanisms at the molecular, cellular, and/or tissue levels (step 4). When the perturbations induced by the toxicant exceed repair capacity or when repair becomes malfunctional, toxicity occurs. Tissue necrosis, cancer, and fibrosis are examples of chemically induced toxicities that follow this four-step course.

Theoretically, the intensity of a toxic effect depends on the concentration and persistence of the ultimate toxicant at its site of action. The ultimate toxicant is the chemical species that reacts with the endogenous target molecule or critically alters the biological environment, initiating structural and/or functional alterations that result in toxicity. The ultimate toxicant can be the original chemical to which the organism is exposed (parent compound), a metabolite or a reactive oxygen or nitrogen species (ROS or RNS) generated during the biotransformation of the toxicant, or an endogenous molecule.

The concentration of the ultimate toxicant at the target molecule depends on the relative effectiveness of the processes that increase or decrease its concentration at the target site (Figure 3–2). Increased concentration is facilitated by absorption, distribution to the site of action, reabsorption, and toxi-cation, while presystemic elimination, distribution away from the site of action, excretion, and detoxication will decrease the toxicant concentration at its target.

Absorption versus Presystemic Elimination

Absorption

The transfer of a chemical from the site of exposure, usually an external or internal body surface, into the systemic circulation is called absorption. Factors that influence absorption include concentration, surface area of exposure, characteristics of the epithelial layer through which the toxicant is being absorbed, and, usually most important, lipid solubility because lipid-soluble molecules are absorbed most easily into cells.

Presystemic Elimination

During transfer from the site of exposure to the systemic circulation, toxicants may be eliminated. This is common for chemicals absorbed from the gastrointestinal (GI) tract because they must first pass through the GI mucosal cells, liver, and lung before being distributed to the rest of the body by the systemic circulation. The GI mucosa and the liver may eliminate a significant fraction of a toxicant during its passage through these tissues. Presystemic or first-pass elimination generally reduces the toxic effects of chemicals that reach their target sites by way of the systemic circulation, but may contribute to injury of the digestive mucosa, the liver, and the lungs because these processes promote toxicant delivery to those sites.

Distribution to and away from the Target

Toxicants exit the blood during the distribution phase, enter the extracellular space, and reach their site or sites of action, usually a macromolecule on either the surface or the interior of a particular type of cell. Chemicals also may be distributed to the site or sites of toxication, usually an intracellular enzyme, where the ultimate toxicant is formed through biotransformation.

Mechanisms Facilitating Distribution to a Target

Porosity of the Capillary Endothelium

Endothelial cells in the hepatic sinusoids and in the renal peritubular capillaries have large fenestrae (50 to 150 nm in diameter) that permit passage of even protein-bound xenobiotics. This favors the accumulation of chemicals in the liver and kidneys.

Specialized Transport Across the Plasma Membrane

Specialized ion channels and membrane transporters can contribute to the delivery of toxicants to intracellular targets. Na+,K+-ATPase, voltage-gated Ca2+ channels, carrier-mediated uptake, endocytosis, and membrane recycling facilitate the entry of toxicants into specific cells, rendering those cells targets. Endocytosis of some toxicant–protein complexes occurs in some cells as well.

Accumulation in Cell Organelles

Amphipathic xenobiotics with a protonatable amine group and lipophilic character accumulate in lysosomes as well as mitochondria. Lysosomal accumulation occurs by pH trapping, that is, diffusion of the amine in unprotonated form into the acidic interior of the organelle, where the amine is protonated, preventing its efflux, so that it impairs phospholipid degradation. Mitochondrial accumulation takes place electrophoretically. The amine is protonated in the intermembrane space and then sucked into the matrix space by the strong negative potential (–220 mV), where it may impair β-oxidation and oxidative phosphorylation.

Reversible Intracellular Binding

Chemicals such as organic and inorganic cations and polycyclic aromatic hydrocarbons accumulate in melanin-containing cells by binding to melanin.

Mechanisms Opposing Distribution to a Target

Binding to Plasma Proteins

Most xenobiotics must dissociate from proteins in order to leave the blood and enter cells. Therefore, strong binding to plasma proteins delays and prolongs the effects and elimination of toxicants.

Specialized Barriers

Brain capillaries lack fenestrae and are joined by extremely tight junctions, preventing the access of hydrophilic chemicals to the brain except by active transport. The spermatogenic cells are surrounded by Sertoli cells that are tightly joined to form the blood–testis barrier. Transfer of hydrophilic toxicants across the placenta is also restricted. However, none of these barriers are effective against lipophilic substances.

Distribution to Storage Sites

Some chemicals accumulate in tissues (i.e., storage sites) where they do not exert significant effects. Such storage decreases toxicant availability for their target sites.

Association with Intracellular Binding Proteins

Binding to nontarget intracellular sites, such as metallothionein, temporarily reduces the concentration of toxicants at the target site.

Export from Cells

Intracellular toxicants may be transported back into the extracellular space. The ATP-dependent membrane transporter family that is known as the multidrug-resistance (mdr) proteins extrudes chemicals from cells.

Excretion versus Reabsorption

Excretion

Excretion is the removal of xenobiotics from blood and their return to the external environment. Excretion is a physical mechanism, whereas biotransformation is a chemical mechanism for eliminating the toxicant.

The route and speed of excretion depend largely on the phys-icochemical properties of the toxicant. The major excretory organs—the kidney and the liver—efficiently remove highly hydrophilic chemicals such as organic acids and bases.

There are no efficient elimination mechanisms for nonvolatile, highly lipophilic chemicals. If resistant to biotransformation, such chemicals are eliminated very slowly and tend to accumulate in the body on repeated exposure. Three rather inefficient processes are available for the elimination of such chemicals: (1) excretion from the mammary gland in breast milk, (2) excretion in bile, and (3) excretion into the intestinal lumen from blood. Volatile, nonreactive toxicants such as gases and volatile liquids diffuse from pulmonary capillaries into the alveoli and are exhaled.

Reabsorption

Toxicants delivered into the renal tubules may diffuse back across the tubular cells into the peritubular capillaries. This tubular fluid reabsorption increases the intratubular concentration as well as the residence time of the chemical by slowing urine flow. Reabsorption by diffusion is dependent on the lipid solubility of the chemical and inversely related to the extent of ionization, because the nonionized molecule is more lipid soluble.

Toxicants delivered to the GI tract by biliary, gastric, and intestinal excretion and secretion by salivary glands and exocrine pancreas may be reabsorbed by diffusion across the intestinal mucosa. Reabsorption of compounds excreted into bile is possible only if they are sufficiently lipophilic or are converted to more lipid-soluble forms in the intestinal lumen.

Toxication versus Detoxication

Toxication

Biotransformation to harmful products is called toxication or metabolic activation. With some xenobiotics, toxication confers physicochemical properties that adversely alter the microenvironment of biological processes or structures. Occasionally, chemicals acquire structural features and reactivity by biotransformation that allows for a more efficient interaction with specific receptors or enzymes. Most often toxication renders xenobiotics and occasionally other molecules in the body, such as nitric oxide, indiscriminately reactive toward endogenous molecules with susceptible functional groups. This increased reactivity may be due to conversion into (1) electrophiles, (2) free radicals, (3) nucleophiles, or (4) redox-active reactants. The most reactive metabolites are electron-deficient molecules and molecular fragments such as electrophiles and neutral or cationic free radicals. Some nucleophiles are reactive (e.g., HCN and CO).

Detoxication

Biotransformations that eliminate the ultimate toxicant or prevent its formation are called detoxications. In some cases, detoxication may compete with toxication.

Detoxication of Toxicants with No Functional Groups

In general, chemicals without functional groups, such as benzene and toluene, are detoxicated in two phases. Initially, a functional group such as hydroxyl or carboxyl is introduced into the molecule, and then, an endogenous acid such as glucuronic acid, sulfuric acid, or an amino acid is added to the functional group by a transferase. With some exceptions, the final products are inactive, highly hydrophilic organic acids that are readily excreted.

Detoxication of Nucleophiles

Nucleophiles generally are detoxicated by conjugation at the nucleophilic functional group, preventing peroxidase-catalyzed conversion of the nucleophiles to free radicals and biotransformation of phenols, aminophenols, catechols, and hydroquinones to electrophilic quinines and quinoneimines.

Detoxication of Electrophiles

Generally, detoxication of electrophilic toxicants involves conjugation with the nucleophile glutathione. This reaction may occur spontaneously or can be facilitated by glutathione S-transferases. Covalent binding of electrophiles to proteins can be regarded as detoxification, provided that the protein has no critical function and does not become a neoantigen or otherwise harmful.

Detoxication of Free Radicals

Because O2•- can be converted into much more reactive compounds (Figure 3–3), its elimination is an important detoxication mechanism. Superoxide dismutases (SODs), located in the cytosol (Cu, Zn-SOD) and the mitochondria (Mn-SOD), convert O2•- to HOOH (Figure 3–4). Subsequently, HOOH is reduced to water by cytosolic glutathione peroxidase or peroxisomal catalase (Figure 3–4).

No enzyme eliminates HO• owing to its extremely short half-life (10-9 s). The only effective protection against HO• is to prevent its formation by converting its precursor, HOOH, to water (Figure 3–4).

Peroxynitrite (ONOO-), which is not a free radical oxidant, is significantly more stable than HO•, and rapidly reacts with CO2 to form reactive free radicals (Figure 3–3). Glutathione peroxidase can reduce ONOO- to nitrite (ONO-). In addition, ONOO- reacts with oxyhemoglobin, heme-containing peroxidases, and albumin, all of which could be important sinks for ONOO-. Furthermore, elimination of the two ONOO-precursors—that is, •NO by reaction with oxyhemoglobin and O2•- by SODs—is a significant mechanism in preventing ONOO- buildup.

Peroxidase-generated free radicals are eliminated by electron transfer from glutathione. This results in the oxidation of glutathione, which is reversed by NADPH-dependent glutathione reductase (Figure 3–5). Thus, glutathione plays an important role in the detoxication of both electrophiles and free radicals.

Detoxication of Protein Toxins

Extra- and intracellular proteases are involved in the inactivation of toxic polypeptides. Venom toxins, such as α- and β-bungaratoxin, erabutoxin, and phospholipase, lose their activity when thioredoxin reduces their essential disulfide bond.

When Detoxication Fails

Detoxication may be insufficient because the toxicant overwhelms the detoxication processes, a reactive toxicant inactivates a detoxicating enzyme, the detoxication is reversed after transfer to other tissues, or harmful byproducts are produced by the detoxication process.

Toxicity is typically mediated by a reaction of the ultimate toxicant with a target molecule (step 2a in Figure 3–1). Subsequently, a series of secondary biochemical events occur, leading to dysfunction or injury that is manifest at various levels of biological organization, such as at the target molecule itself, cell organelles, cells, tissues and organs, and even the whole organism.

Attributes of Target Molecules

Practically all endogenous compounds are potential targets for toxicants. The most prevalent and toxicologically relevant targets are nucleic acids (especially DNA), proteins, and membranes. The first target for reactive metabolites is often the enzyme responsible for their production or the adjacent intracellular structures. Not all targets for chemicals contribute harmful effects. Covalent binding to proteins without adverse consequences may even represent a form of detoxication by sparing toxicologically relevant targets. To conclusively identify a target molecule as being responsible for toxicity, it should be demonstrated that the ultimate toxicant (1) reacts with the target and adversely affects its function, (2) reaches an effective concentration at the target site, and (3) alters the target in a way that is mechanistically related to the observed toxicity.

Types of Reactions

The ultimate toxicant may bind to the target molecules noncovalently or covalently and may alter it by hydrogen abstraction, electron transfer, or enzymatically.

Noncovalent Binding

Apolar interactions or the formation of hydrogen and ionic bonds is typically involved in the interaction of toxicants with targets such as membrane receptors, intracellular receptors, ion channels, and some enzymes. Noncovalent binding usually is reversible because of the comparatively low bonding energy.

Covalent Binding

Being practically irreversible, covalent binding permanently alters endogenous molecules. Covalent adduct formation is common with electrophilic toxicants such as nonionic and cationic electrophiles and radical cations. These toxicants react with nucleophilic atoms that are abundant in biological macromolecules, such as proteins and nucleic acids. Neutral free radicals such as HO•, •NO2, and Cl3C• also can bind covalently to biomolecules.

Hydrogen Abstraction

Neutral free radicals can readily abstract H atoms from endogenous compounds, converting those compounds into radicals. Radicals can also remove hydrogen from CH2 groups of free amino acids or from amino acid residues in proteins and convert them to carbonyls, forming cross-links with DNA or other proteins.

Electron Transfer

Chemicals can exchange electrons to oxidize or reduce other molecules, leading to formation of harmful byproducts. For example, chemicals can oxidize Fe(II) in hemoglobin to Fe(III), producing methemoglobinemia.

Enzymatic Reactions

A few toxins act enzymatically on specific target proteins. For example, diphtheria toxin blocks the function of elongation factor 2 in protein synthesis and cholera toxin activates a G protein through such a mechanism.

In summary, most ultimate toxicants act on endogenous molecules on the basis of their chemical reactivity. Those with more than one type of reactivity may react by different mechanisms with various target molecules.

Effects of Toxicants on Target Molecules

Dysfunction of Target Molecules

Some toxicants activate protein target molecules, mimicking endogenous ligands. More commonly, chemicals inhibit the function of target molecules, by blocking neurotransmitter receptors or ion channels, inhibiting enzymes, and interfering with cytoskeleton dynamics.

Protein function is impaired when conformation or structure is altered by interaction with the toxicant. Many proteins possess critical moieties that are essential for catalytic activity or assembly to macromolecular complexes. Covalent and/or oxidative modification of these moieties by xenobiotics can cause aberrant signal transduction and/or impaired maintenance of the cell's energy and metabolic homeostasis. Toxicants may also interfere with the template function of DNA. The covalent binding of chemicals to DNA causes nucleotide mispairing during replication.

Destruction of Target Molecules

In addition to adduct formation, toxicants alter the primary structure of endogenous molecules by means of cross-linking and fragmentation. Cross-linking imposes both structural and functional constraints on the linked molecules.

Other target molecules are susceptible to spontaneous degradation after chemical attack. Free radicals such as Cl3COO• and HO• can initiate peroxidative degradation of lipids by hydrogen abstraction from fatty acids, thereby destroying lipids in cellular membranes and also generating endogenous lipid radicals and electrophiles, which can harm the structure of DNA. Several forms of DNA fragmentation are caused by toxicants, for example, multiple hydroxyl radical attacks on a short length of DNA, which occur after ionizing radiation, causing double-strand breaks that are typically lethal to the affected cell.

Neoantigen Formation

Covalent binding of xenobiotics or their metabolites to protein may evoke an immune response. Some chemicals (e.g., dinitrochlorobenzene, penicillin, and nickel) bind to proteins spontaneously. Others may obtain reactivity by autooxidation to quinones (e.g., urushiols, the allergens in poison ivy) or by enzymatic biotransformation.

Toxicity Not Initiated by Reaction with Target Molecules

Some xenobiotics alter the biological microenvironment (see step 2b in Figure 3–1) leading to a toxic response. Included here are (1) chemicals that alter H+ ion concentrations in the aqueous biophase, (2) solvents and detergents that physicochemically alter the lipid phase of cell membranes and destroy transmembrane solute gradients, and (3) xenobiotics that cause harm merely by occupying a site or space.

Reaction of toxicants with a target molecule may result in impaired cellular function as the third step in the development of toxicity (Figures 3–1 and 3–6). Each cell in a multicellular organism carries out defined programs, some of which determine whether cells undergo division, differentiation, or apoptosis. Other programs control the ongoing (momentary) activity of differentiated cells. For regulation of these cellular programs, cells possess signaling networks that can be activated and inactivated by external signaling molecules.

As outlined in Figure 3–6, the nature of the primary cellular dysfunction caused by toxicants, but not necessarily the ultimate outcome, depends on the role of the target molecule affected. The reaction of a toxicant with targets serving external functions can influence the operation of other cells and integrated organ systems. However, if the target molecule is involved predominantly in the cell's internal maintenance, the resultant dysfunction can ultimately compromise survival of the cell.

Toxicant-Induced Cellular Dysregulation

Cells are regulated by signaling molecules that activate specific cellular receptors linked to signal-transducing networks that transmit the signals to the regulatory regions of genes and/or functional proteins. Receptor activation may ultimately lead to altered gene expression and/or a chemical modification of specific proteins, typically by phosphorylation. Programs controlling the destiny of cells primarily affect gene expression, whereas those regulating the ongoing activities primarily influence the activity of functional proteins. However, one signal often evokes both responses because of branching and interconnection of signaling networks.

Dysregulation of Gene Expression

Dysregulation of gene expression may occur at elements that are directly responsible for transcription, at components of the intracellular signal-transduction pathway, and at the synthesis, storage, or release of the extracellular signaling molecules.

Dysregulation of Transcription

Transcription of genetic information from DNA to mRNA is controlled largely by interplay between transcription factors (TFs) and the regulatory or promoter region of genes. Xenobiotics may interact with the promoter region of the gene, the TFs, or other components of the transcription initiation complex. However, altered activation of TFs appears to be the most common modality.

Many natural compounds, such as hormones and vitamins, influence gene expression by binding to and activating TFs. Whereas xenobiotics may mimic the natural ligands, both may cause toxicity when administered at extreme doses or at critical periods during ontogenesis. Other compounds that act on TFs can also change the pattern of cell differentiation by overex-pressing various genes.

Xenobiotics may also dysregulate transcription by altering the regulatory gene regions and promoter methylation.

Dysregulation of Signal Transduction

Extracellular signaling molecules, such as growth factors, cytokines, hormones, and neurotransmitters, can ultimately activate TFs utilizing cell surface receptors and intracellular signal-transducing networks. Figure 3–7 depicts such networks and identifies some important signal-activated TFs that control transcriptional activity of genes that influence cell cycle progression and thus determine the fate of cells. An example is the c-Myc protein, which, on dimerizing with Max protein and binding to its cognate nucleotide sequence, transactivates cyclin D and E genes. The cyclins, in turn, accelerate the cell-division cycle by activating cyclin-dependent protein kinases, which are involved in regulating the cell cycle. Mitogenic signaling molecules thus induce cellular proliferation.

The signal from the cell surface receptors to the TFs is relayed by successive protein–protein interactions and protein phosphorylations, that is, a signal molecule phosphorylates another protein like mitogen-activated kinase (MAPK), which activates that protein to phosphorylate and activate another. For example, ligands induce growth factor receptors (item 6 in Figure 3–7) on the surface of all cells to self-phosphorylate, and these phosphorylated receptors then bind to adapter proteins through which they activate Ras. The active Ras initiates the MAPK cascade, involving serial phosphorylations of protein kinases, which finally reaches the TFs. These signal transducers are typically but not always activated by phosphorylation catalyzed by protein kinases and are usually inactivated by dephosphorylation carried out by protein phosphatases.

Chemicals may cause aberrant signal transduction most often by altering protein phosphorylation, and occasionally by interfering with the GTPase activity of G proteins, disrupting normal protein–protein interactions, or establishing abnormal ones, or by altering the synthesis or degradation of signaling proteins. Such interventions may ultimately influence cell cycle progression.

Chemically Altered Signal Transduction with Proliferative Effect: Xenobiotics that facilitate phosphorylation of signal transducers often promote mitosis and tumor formation. The phorbol esters and fumonisin B activate protein kinase C (PKC) mimicking diacylglycerol (DAG), one of the physiologic activators of PKC (Figure 3–7). The other physiologic PKC activator, Ca2+, is mimicked by Pb2+. Activated PKC promotes mitogenic signaling by starting a cascade that activates other kinases and allows certain TFs to bind to DNA. Protein kinases may also be activated by interacting proteins that had been altered by a xenobiotic.

Aberrant phosphorylation of proteins may result from decreased dephosphorylation by phosphatases. Inhibition of phosphatases appears to be the underlying mechanism of the mitogenic effect of various chemicals, oxidative stress, and ultraviolet (UV) irradiation. Soluble protein phosphatase 2A (PP2A) in cells is likely responsible for reversing the growth factor-induced stimulation of MAPK, thereby controlling the extent and duration of MAPK activity under control. PP2A also removes an activating phosphate from a mitosis-triggering protein kinase. Several natural toxins are extremely potent inhibitors of PP2A, including the blue-green algae poison micro-cystin-LR and the dinoflagellate-derived okadaic acid.

Apart from phosphatases, other inhibitory binding proteins can keep signaling under control. For example, IκB, which binds to NF-κB, preventing its transfer into the nucleus and its function as a TF. On phosphorylation, IκB becomes degraded and NF-κB is set free. NF-κB is an important contributor to proliferative and prolife signaling, as well as inflammation and acute-phase reactions. IκB degradation and NF-κB activation can also be induced by oxidative stress.

Chemically Altered Signal Transduction with Antiproliferative Effect: Downturning of increased proliferative signaling after cell injury may compromise replacement of injured cells (follow the path in Figure 3–7: inhibition of Raf → diminished degradation of IκB → diminished binding of NF-κB to DNA → diminished expression of c-Myc mRNA). Down-regulation of a normal mitogenic signal is a step away from survival and toward apoptosis.

Dysregulation of Extracellular Signal Production

Hormones of the anterior pituitary exert mitogenic effects on endocrine glands in the periphery by acting on cell surface receptors. Pituitary hormone production is under negative feedback control by hormones of the peripheral glands. Perturbation of this circuit adversely affects pituitary hormone secretion and, in turn, the peripheral glands. Decreased secretion of pituitary hormone produces apoptosis followed by involution of the peripheral target gland.

Dysregulation of Ongoing Cellular Activity

Toxicants can adversely affect ongoing cellular activity in specialized cells by disrupting any step in signal coupling.

Dysregulation of Electrically Excitable Cells

Many xenobi-otics influence cellular activity in excitable cells, such as neurons, skeletal, cardiac, and smooth muscle cells. Release of neurotransmitters and muscle contraction are controlled by transmitters and modulators synthesized and released by adjacent neurons. Chemicals that interfere with these mechanisms are listed in Table 3–1.

Perturbation of ongoing cellular activity by chemicals may be due to an alteration in (1) the concentration of neurotransmitters, (2) receptor function, (3) intracellular signal transduction, or (4) the signal-terminating processes.

Alteration in Neurotransmitter Levels: Chemicals may alter synaptic levels of neurotransmitters by interfering with their synthesis, storage, release, or removal from the vicinity of the receptor.

Toxicant–Neurotransmitter Receptor Interactions: Some chemicals interact directly with neurotransmitter receptors, including (1) agonists that associate with the ligand-binding site on the receptor and mimic the natural ligand, (2) antagonists that occupy the ligand-binding site but cannot activate the receptor, (3) activators, and (4) inhibitors that bind to a site on the receptor that is not directly involved in ligand binding. In the absence of other actions, agonists and activators mimic, whereas antagonists and inhibitors block, the physiologic responses characteristic of endogenous ligands. Because there are multiple types of receptors for each neurotransmitter, these receptors may be affected differentially by toxicants.

Toxicant–Signal Transducer Interactions: Many chemicals alter neuronal and/or muscle activity by acting on signal-transduction processes. Voltage-gated Na+ channels, which transduce and amplify excitatory signals generated by ligand-gated cation channels, are activated or inactivated by several toxins (see Table 3–1).

Toxicant–Signal Terminator Interactions: The cellular signal generated by cation influx is terminated by removal of the cations through channels or by transporters. Inhibition of cation export may prolong excitation.

Dysregulation of the Activity of Other Cells

Whereas many signaling mechanisms operate in nonexcitable cells, such as exocrine secretory cells, Kupffer cells, and pancreatic beta cells, disturbance of these processes is usually less consequential.

Impairment of Internal Cellular Maintenance: Mechanisms of Toxic Cell Death

For survival, all cells must synthesize endogenous molecules, assemble macromolecular complexes, membranes, and cell or-ganelles, maintain the intracellular environment, and produce energy for operation. Agents that disrupt these functions jeopardize survival. There are three critical biochemical disorders that chemicals inflicting cell death may initiate, namely, ATP depletion, sustained rise in intracellular Ca2+, and overproduction of ROS and RNS.

Depletion of ATP

ATP plays a central role in cellular maintenance both as a chemical for biosynthesis and as the major source of energy. ATP is utilized in numerous biosynthetic reactions, and is incorporated into cofactors as well as nucleic acids. It is required for muscle contraction and polymerization of the cytoskeleton, fueling cellular motility, cell division, vesicular transport, and the maintenance of cell morphology. ATP drives ion transporters (e.g., Na+,K+-ATPase) that maintain conditions essential for various cell functions.

Chemical energy is released by hydrolysis of ATP to ADP or AMP. The ADP is rephosphorylated in the mitochondria by ATP synthase (Figure 3–8) via a process that couples oxidation of hydrogen to water and is termed oxidative phosphorylation.

Oxidative phosphorylation also requires several steps, each of which can be interfered with by toxins, as described in Table 3–2. Impairment of oxidative phosphorylation is detrimental to cells because failure of ADP rephosphorylation results in the accumulation of ADP and its breakdown products, as well as depletion of ATP.

Substances in class A interfere with the delivery of hydrogen to the electron transport chain. Class B chemicals inhibit the transfer of electrons along the electron transport chain to oxygen. Class C agents interfere with oxygen delivery to the terminal electron transporter, cytochrome oxidase. Chemicals in class D inhibit oxidative phosphorylation by: (1) direct inhibition of ATP synthase, (2) interference with ADP delivery, (3) interference with inorganic phosphate delivery, and (4) deprivation of ATP synthase from its driving force, the controlled influx of protons into the matrix space. Finally, chemicals causing mitochondrial DNA injury, thereby impairing synthesis of specific proteins encoded by the mitochondrial genome, are listed in group E.

Sustained Rise of Intracellular Ca2+

Intracellular Ca2+ levels are highly regulated and maintained by the impermeability of the plasma membrane to Ca2+ and by transport mechanisms that remove Ca2+ from the cytoplasm. Ca2+ is actively pumped from the cytosol across the plasma membrane and is sequestered in the endoplasmic reticulum and mitochondria (Figure 3–8).

Toxicants induce elevation of cytoplasmic Ca2+ levels by promoting Ca2+ influx into or inhibiting Ca2+ efflux from the cytoplasm (Table 3–3). Opening of the ligand- or voltage-gated Ca2+ channels or damage to the plasma membrane causes Ca2+ to move down its concentration gradient from extracellular fluid to the cytoplasm. Toxicants also may increase cytosolic Ca2+ inducing its leakage from the mitochondria or the endoplasmic reticulum. They also may diminish Ca2+ efflux through inhibition of Ca2+ transporters or depletion of their driving forces. Sustained elevation of intracellular Ca2+ is harmful because it can result in (1) depletion of energy reserves by inhibiting the ATPase used in oxidative phosphorylation, (2) dysfunction of microfilaments, (3) activation of hydrolytic enzymes, and (4) generation of ROS and RNS.

There are at least three mechanisms by which sustained elevations in intracellular Ca2+ levels influence the cellular energy balance. First, high cytoplasmic Ca2+ levels cause increased mitochondrial Ca2+ uptake by the Ca2+ “uniporter,” which, like ATP synthase, utilizes the inside negative mitochondrial membrane potential as the driving force. Consequently, mitochondrial Ca2+ uptake dissipates the membrane potential and inhibits the synthesis of ATP. Moreover, agents that oxidize mitochondrial NADH activate a transporter that extrudes Ca2+ from the matrix space. The ensuing continuous Ca2+ uptake and export (“Ca2+ cycling”) by the mitochondria further compromise oxidative phosphorylation.

Second, an uncontrolled rise in cytoplasmic Ca2+ causes cell injury by microfilamental dissociation. An increase of cyto-plasmic Ca2+ causes dissociation of actin filaments from proteins that promote anchoring of the filament to the plasma membrane, predisposing the membrane to rupture.

Third, high Ca2+ levels may lead to activation of hydrolytic enzymes that degrade proteins, phospholipids, and nucleic acids. Many integral membrane proteins are targets for Ca2+-activated neutral proteases, or calpains. Indiscriminate activation of phospholipases by Ca2+ causes membrane breakdown directly and by the generation of detergents. Activation of a Ca2+–Mg2+-dependent endonuclease causes fragmentation of chromatin.

Overproduction of ROS and RNS

A number of xenobiotics can directly generate ROS and RNS, such as the redox cyclers and transition metals (Figure 3–3). Overproduction of ROS and RNS can be secondary to intracellular hypercalcemia, as Ca2+ helps generate ROS and/or RNS by activating dehydrogenases in the citric acid cycle leading to increased activity in the electron transport chain and increased formation of O2•- and HOOH, and by activating nitric oxide synthase, which leads to formation of ONOO-.

Interplay between the Primary Metabolic Disorders Spells Cellular Disaster

The primary derailments in cellular biochemistry discussed above may interact and amplify each other in a number of ways:

1. Depletion of cellular ATP reserves deprives the endoplasmic and plasma membrane Ca2+ pumps of fuel, causing elevation of Ca2+ in the cytoplasm. With the influx of Ca2+ into the mitochondria, the mitochondrial membrane potential declines, hindering ATP synthase.

2. Intracellular hypercalcemia facilitates formation of ROS and RNS, which oxidatively inactivates the Ca2+ pump, aggravating the hypercalcemia.

3. ROS and RNS can also drain the ATP reserves. •NO is a reversible inhibitor of cytochrome oxidase, NO+ (nitroso-nium cation, a product of •NO) inactivates glyceraldehyde 3-phosphate dehydrogenase and impairs glycolysis, whereas ONOO- irreversibly inactivates several components of the electron transport chain, inhibiting cellular ATP synthesis.

4. Furthermore, ONOO- can induce DNA single-strand breaks, which activate poly(ADP-ribose) polymerase (PARP). As part of the repair strategy, activated PARP transfers multiple ADP-ribose moieties from NAD+ to nuclear proteins and PARP itself. Because consumption of NAD+ severely compromises ATP synthesis (see Figure 3–8) and resynthesis of NAD+ consumes ATP, a cellular energy deffcit occurs as a major consequence of DNA damage by ONOO-.

Mitochondrial Permeability Transition (MPT) and the Worst Outcome: Necrosis

Mitochrondrial Ca2+ uptake, decreased mitochondrial membrane potential, generation of ROS and RNS, depletion of ATP, and consequences of the primary metabolic disorders (e.g., accumulation of inorganic phosphate, free fatty acids, and lysophosphatides) are all considered as causative factors of an abrupt increase in the mitochondrial inner-membrane permeability, termed MPT. This is believed to be caused by the opening of a proteinaceous pore that spans both mitochondrial membranes and is permeable to solutes of 1500 Da. This opening permits free influx into the matrix space of protons, causing rapid and complete dissipation of the membrane potential, cessation of ATP synthesis, and the osmotic influx of water causing mitochondrial swelling. Ca2+ accumulated in the matrix space effluxes through the pore, flooding the cytoplasm. Such mitochondria are not only incapable of synthesizing ATP, but also even waste the remaining sources because depolarization of the inner membrane forces the ATP synthase to operate in the reverse mode, as an ATPase, hydrolyzing ATP. Then glycolysis may become compromised by the insufficient ATP supply to the glycolytic enzymes that require ATP (hexokinase and phosphofructokinase). A complete bioenergetic catastrophe ensues in the cell if the metabolic disorders evoked by the toxicant (such as those listed in Tables 3–2 and 3–3) are so extensive that most or all mitochondria undergo MPT, causing depletion of cellular ATP, and culminating in cell lysis or necrosis (see Figure 3–9).

An Alternative Outcome of MPT: Apoptosis

Chemicals that adversely affect cellular energy metabolism, Ca2+ homeostasis, and redox state and ultimately cause necrosis may also induce apoptosis. While the necrotic cell swells and lyses, the apoptotic cell shrinks; its nuclear and cytoplasmic materials condense, and then it breaks into membrane-bound fragments (apoptotic bodies) that are phagocytosed.

In contrast to the random sequence of multiple metabolic defects that a cell suffers on its way to necrosis, the routes to apoptosis are ordered, involving cascade-like activation of catabolic processes that finally disassemble the cell. Many details of the apoptotic pathways are presented schematically in Figure 3–10. It appears that most, if not all, chemical-induced cell deaths will involve the mitochondria, and that MPT is a crucial event. Another related event is release into the cytoplasm of cytochrome c (cyt c), a small hemeprotein that normally resides in the mitochondrial intermembrane space attached to the surface of inner membrane.

As cyt c is the penultimate link in the mitochondrial electron transport chain, its loss will block ATP synthesis, increase for-mation of O2• -, and potentially thrust the cell toward necrosis. Simultaneously, the unleashed cyt c represents a signal or an initial link in the chain of events directing the cell to the apoptotic path (Figure 3–10). On binding, together with ATP, to an adapter protein, cyt c can induce proteolytic cleavage of proteins called caspases or cysteineproteases that cleave cytoplasmic proteins into fragments, beginning apoptosis. Some caspases (e.g., 2, 8, and 9) activate procaspases. These signaling caspases carry the activation wave to the so-called effector caspases (e.g., 3, 6, and 7), which activate or inactivate specific cellular proteins.

The decisive mitochondrial events of cell death are controlled by the Bcl-2 family of proteins, which includes members that facilitate (e.g., Bax, Bad, and Bid) and those that inhibit (e.g., Bcl-2 and Bcl-XL) these processes. Death-promoting members can oligomerize and form pores in the mitochondrial outer membrane, thereby facilitating release of cyt c and other inter-membrane proapoptotic proteins via MPT induced by toxic insult of the mitochondria; however, mitochondrial outer membrane permeabilization (MOMP) alone by Bax and its congeners is sufficient to evoke egress of cyt c from the mitochondria. The relative amount of these antagonistic proteins functions as a regulatory switch between cell survival and death.

The proapoptotic Bax and Bid proteins also represent links whereby death programs, initiated by DNA damage in the nucleus or by stimulation of certain receptors called Fas receptors at the cell surface, can trigger the mitochondria into the apoptotic process (Figure 3–10). DNA damage induces stabilization and activation of p53 protein, which increases expression of Bax protein, a member of the Bcl-2 family. DNA damage is potentially mutagenic and carcinogenic and apoptosis of cells with damaged DNA is an important self-defense against oncogenesis. Stimulation of Fas receptors can directly activate caspases, and can engage the mitochondria into the death program via caspase-mediated activation of Bid, another member of the Bcl-2 family (Figure 3–10). Thus, apoptosis can be executed via multiple pathways; the preferred route will depend on the initial insult as well as on the type and state of the cell.

ATP Availability Determines the Form of Cell Death

Many xenobiotics can cause both apoptosis and necrosis. Toxicants tend to induce apoptosis at low exposure levels or early after exposure at high levels, whereas they cause necrosis later at high exposure levels. Recent findings suggest that the availability of ATP is critical in determining the form of cell death. When only a few mitochondria develop MPT, they, and with them the proapoptotic signals (e.g., externalized cyt c), are removed by lysosomal autophagy. When MPT involves more mitochondria, the autophagic mechanism becomes overwhelmed and the released cyt c initiates caspase activation and apoptosis (Figure 3–10). When MPT involves virtually all mitochondria, ATP becomes severely depleted, preventing execution of the ATP-requiring apoptotic program and cytolysis occurs.

The fourth step in the development of toxicity is inappropriate repair (Figure 3–1). Many toxicants alter macromolecules, which, if not repaired, cause damage at higher levels of the biological hierarchy in the organism and influence the progression of toxicity.

Molecular Repair

Damaged molecules may be repaired in different ways. Some chemical alterations, such as oxidation of protein thiols and methylation of DNA, are simply reversed. Hydrolytic removal of the molecule's damaged unit or units and insertion of a newly synthesized unit or units often occur with chemically altered DNA and peroxidized lipids. In some instances, the damaged molecule is totally degraded and resynthesized.

Repair of Proteins

Thiol groups are essential for the function of numerous proteins. Oxidation of protein thiols can be reversed by enzymatic reduction that is catalyzed by thioredoxin and glutaredoxin. Once oxidized, the catalytic thiol groups in these proteins are recycled by reduction with NADPH.

Repair of oxidized hemoglobin (methemoglobin) occurs by means of electron transfer from cytochrome b5, which is then regenerated by a NADH-dependent cytochrome b5 reductase. Molecular chaperones such as the heat-shock proteins are synthesized in large quantities in response to protein denaturation. Damaged proteins can either be refolded with the help of chaperone proteins or degraded, following ubiquitination in proteasomes. Also, the ATP/ubiquitin-dependent proteolytic system is specialized in controlling the level of regulatory proteins (e.g., p53, IκB, and cyclins) that eliminate damaged or mutated intracellular proteins. Additionally, proteins can be eliminated by proteolysis in lysosomes.

Repair of Lipids

Peroxidized lipids are repaired by a complex process involving a series of reductants, glutathione peroxidase, and glutathione reductase. NADPH is needed to recycle the reductants that are oxidized in the process.

Repair of DNA

Despite its high reactivity with electrophiles and free radicals, nuclear DNA is remarkably stable, in part because it is packaged in chromatin and because several repair mechanisms are available to correct alterations. Mitochondrial DNA, however, lacks histones and efficient repair mechanisms and therefore is more prone to damage.

Direct Repair

Certain covalent DNA modifications are directly reversed by enzymes such as DNA photolyase, which cleaves adjacent pyrimidines dimerized by UV light. This chromophore-equipped enzyme functions only in light-exposed cells. Minor adducts, such as methyl groups, attached to the O6 position of guanine are removed by O6-alkylguanine-DNA-alkyltransferase.

Excision Repair

Base excision and nucleotide excision are two mechanisms for removing damaged bases from DNA (Chapters 8 and 9). Lesions that do not cause major distortion of the helix are removed typically by base excision, in which the altered base is recognized by a relatively substrate-specific DNA-glycosylase that hydrolyzes the N-glycosidic bond, releasing the modified base and creating an apurinic or apyrimidinic (AP) site in the DNA. The AP site is recognized by the AP endonuclease, which hydrolyzes the phosphodiester bond adjacent to the abasic site. After its removal, the abasic sugar is replaced with the correct nucleotide by a DNA polymerase and is sealed in place by a DNA ligase.

Bulky adducts are removed by nucleotide-excision repair. An ATP-dependent nuclease recognizes the distorted double helix and excises a number of intact nucleotides on both sides of the lesion together with the one containing the adduct. The excised section of the strand is restored by insertion of nucleotides into the gap by DNA polymerase and ligase, using the complementary strand as a template.

PARP appears to be an important contributor in excision repair. On base damage or single-strand break, PARP binds to the injured DNA and becomes activated. The active PARP cleaves NAD+ to use the ADP-ribose moiety of this cofactor for attaching long chains of polymeric ADP-ribose to nuclear proteins. This causes the DNA to unwind, giving access to the repair enzymes and allowing the broken DNA to be fixed.

Recombinational (or Postreplication) Repair

Recombinational repair occurs when the excision of a bulky adduct or an intrastrand pyrimidine dimer fails to occur before DNA replication begins. At replication, such a lesion prevents DNA polymerase from polymerizing a daughter strand along a sizable stretch of damaged parent strand. Replication results in two homologous (“sister”) yet dissimilar DNA duplexes: one that has a large postreplication gap in its daughter strand and an intact duplex synthesized at the opposite leg of the replication fork. This intact sister duplex completes the postreplication gap in the damaged sister duplex by recombination (“crossover”) of the appropriate strands of the two homologous duplexes. After separation, the sister duplex that originally contained the gap carries in its daughter strand a section originating from the parent strand of the intact sister, which in turn carries in its parent strand a section originating from the daughter strand of the damaged sister—a process of sister chromatid exchange. A combination of excision and recombinational repairs occurs in restoration of DNA with interstrand cross-links.

Cellular Repair: A Strategy in Peripheral Neurons

Repair of damaged neurons is minimally applied in overcoming cellular injuries because mature neurons have lost their ability to multiply. In peripheral neurons with axonal damage, repair does occur and requires macrophages and Schwann cells. Macrophages remove debris by phagocytosis and produce cytokines and growth factors, which activate Schwann cells to proliferate and transdifferentiate into a growth-supporting mode. While comigrating with the regrowing axon, Schwann cells physically guide as well as chemically lure the axon to reinnervate the target cell.

In the mammalian central nervous system, axonal regrowth is prevented by growth inhibitory glycoproteins and chondroitin sulfate proteoglycans produced by the oligodendrocytes and by the scar produced by astrocytes. Although damage to central neurons is irreversible, the large number of reserve nerve cells can partly compensate by taking over the functions of lost neurons.

Tissue Repair

In tissues with cells capable of multiplying, damage is reversed by apoptosis or necrosis of the injured cells and regeneration of the tissue by proliferation.

Apoptosis: An Active Deletion of Damaged Cells

Apoptosis initiated by cell injury can be regarded as tissue repair. A cell undergoing apoptosis shrinks as its nuclear and cytoplasmic materials condense, and then it breaks into membrane-bound fragments (apoptotic bodies) that are phagocytosed without inflammation. Also, apoptosis may intercept the process leading to neoplasia by eliminating the cells with potentially mutagenic DNA damage.

Apoptosis of damaged cells has a full value as a tissue repair process only for tissues that are made up of constantly renewing cells (e.g., the bone marrow, the respiratory and GI epithelium, and the epidermis of the skin), or of conditionally dividing cells (e.g., hepatic and renal parenchymal cells), because the apoptotic cells are readily replaced. The value of apoptosis as a tissue repair strategy is markedly lessened in organs containing non-replicating and nonreplaceable cells, such as the neurons, cardiac muscle cells, and female germ cells.

Proliferation: Regeneration of Tissue

Tissues are composed of various cells and the extracellular matrix. Cadherins allow adjacent cells to adhere to one other, whereas connexins connect neighboring cells internally by association of these proteins into gap junctions. Integrins link cells to the extracellular matrix. Therefore, repair of injured tissues involves both regeneration of lost cells and the extracellular matrix and reintegration of the newly formed elements into tissues and organs.

Replacement of Lost Cells by Mitosis

Soon after injury, cells adjacent to the damaged area enter the cell-division cycle. Quiescent cells residing in G0 enter G1 and progress to mitosis (M).

Sequential changes in gene expression occur in the cells that are destined to divide. Early after injury, intracellular signaling turns on, and expression of numerous genes is increased. Among these so-called immediate-early genes are those that code for TFs that amplify the initial gene-activation process by stimulating other genes directly or through cell surface receptors and the coupled transducing networks. A few hours later, the so-called delayed-early genes are expressed whose products regulate the cell-division cycle. Genes for the cell cycle accelerator proteins and also genes whose products decelerate the cell cycle become temporarily overexpressed, suggesting that this duality keeps tissue regeneration precisely regulated. Thus, genetic expression is reprogrammed so that DNA synthesis and mitosis gain priority over specialized cellular activities.

The regenerative process is probably initiated by the release of chemical mediators from damaged cells. Nonparenchymal cells, such as resident macrophages and endothelial cells, are receptive to these chemical signals and produce a host of signaling molecules that promote and propagate the regenerative process. The cytokinesTNF-α and interleukin=6 (IL-6) purportedly promote transition of the quiescent cells into cell cycle (“priming”), whereas the growth factors, especially the hepatocyte growth factor (HGF) and transforming growth factor-α (TGF-α), initiate the progression of the “primed” cells in the cycle toward mitosis.

Besides mitosis, cell migration also significantly contributes to restitution of certain tissues. In the mucosa of the GI tract, cells of the residual epithelium rapidly migrate to the site of injury as well as elongate and thin to reestablish the continuity of the surface even before this could be achieved by cell replication. Mucosal repair is dictated by growth factors and cytokines operative in tissue repair elsewhere and also by specific peptides associated with the mucous layer of the GI tract that become overexpressed at sites of mucosal injury.

Replacement of the Extracellular Matrix

The extracellular matrix is composed of proteins, glycosaminoglycans, and the glycoprotein and proteoglycans. Activation of resting stellate cells is mediated chiefly by two growth factors, platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β), that may be released from platelets accumulating and degranulating at sites of injury and later from the activated stellate cells themselves. Proliferation of stellate cells is induced by the potent mitogen PDGF, whereas TGF-β acts on the stellate cells to stimulate the synthesis of extracellular matrix components, including collagens, fibronectin, tenascin, and proteoglycans. TGF-β also plays a central role in extracellular matrix formation in other tissues.

Side Reactions to Tissue Injury

In addition to mediators that aid in the replacement of lost cells and the extracellular matrix, resident macrophages and endothelial cells activated by cell injury also produce inflammation, altered production of acute-phase protein, and generalized reactions such as fever.

Inflammation

Cells and Mediators: Alteration of the microcirculation and accumulation of inflammatory cells are largely initiated by resident macrophages secreting cytokines such as TNF-α and IL-1 in response to tissue damage. These cytokines, in turn, stimulate neighboring stromal cells, such as the endothelial cells and fibroblasts, to release mediators that induce dilation of the local microvasculature and cause permeabilization of capillaries. Activated endothelial cells also facilitate the egress of circulating leukocytes into the injured tissue by releasing chemoattractants and expressing cell-adhesion molecules. Subsequently a stronger interaction (adhesion) is established between the endothelial cells and leukocytes with participation of intercellular adhesion molecules (e.g., ICAM-1) and leukocytes are able to enter the tissues by crossing the endothelial layer. This is facilitated by gradients of chemoattractants, including chemotactic cytokines, platelet-activating factor (PAF) and leukotriene B4, that induce expression of leukocyte integrins.

Inflammation Produces Reactive Oxygen and Nitrogen Species: Macrophages as well as leukocytes recruited to a site of injury undergo a respiratory burst, producing free radicals and activated enzymes. Membrane-bound NAD(P)H oxidase, activated in both macrophages and granulocytes, produces superoxide anion radical (O2•-) from molecular oxygen. The O2•- can give rise to the hydroxyl radical (HO•).

Macrophages, but not granulocytes, generate another cytotoxic free radical, nitric oxide (•NO), from arginine by nitric oxide synthase:

Subsequently, O2•- and •NO, both of which are products of activated macrophages, can react with each other, yielding peroxynitrite anion; on reaction with carbon dioxide, this decays into two radicals, nitrogen dioxide and carbonate anion radical:

Granulocytes discharge the lysosomal enzyme myeloperoxidase into engulfed extracellular spaces, the phagocytic vacuoles.

Myeloperoxidase catalyzes the formation of hypochlorous acid (HOCl) from hydrogen peroxide (HOOH) and chloride ion:

HOCl can form HO• as a result of electron transfer from Fe2+ or from O2•- to HOCl:

All the above reactive chemicals, as well as the discharged lysosomal proteases, are destructive products of inflammatory cells. Although these chemicals exert antimicrobial activity at the site of microbial invasion, they can damage adjacent healthy tissues at the site of toxic injury and contribute to propagation of tissue injury.

Altered Protein Synthesis: Acute-Phase Proteins

Cytokines released from macrophages and endothelial cells of injured tissues, IL-6, IL-1, and TNF, act on cell surface receptors to increase or decrease the transcriptional activity of genes encoding certain proteins called positive and negative acute-phase proteins.

Positive acute-phase proteins may play roles in minimizing tissue injury and facilitating repair. For example, many of them inhibit lysosomal proteases released from the injured cells and recruited leukocytes.

Because negative acute-phase proteins play important roles in the toxication and detoxication of xenobiotics, the disposition and toxicity of chemicals may be altered markedly during the acute phase of tissue injury.

Generalized Reactions

Cytokines released from activated macrophages and endothelial cells at the site of injury also may evoke neurohormonal responses. Thus, IL-1, TNF, and IL-6 alter the temperature set point of the hypothalamus, triggering fever. In addition, IL-1 and IL-6 act on the pituitary to induce the release of ACTH, which in turn stimulates the secretion of cortisol from the adrenals. This represents a negative feedback loop because corticosteroids inhibit cytokinegene expression.

Mechanisms of Adaptation

Adaptation involves responses acting to preserve or regain the biological homeostasis in the face of increased harm from a noxious stimulus. Adaptation of toxicity may result from biological changes causing (1) diminished delivery of the toxicant to the target, (2) decreased size or susceptibility of the target, (3) increased capacity of the organism to repair itself, and (4) strengthened mechanisms to compensate the toxicant-inflicted dysfunction. Adaptation involves sensing the noxious chemical and/or the initial damage or dysfunction, and a response that typically occurs through altered gene expression.

Certain chemicals induce adaptive changes that lessen their delivery by diminishing the absorption, increasing their sequestration by intracellular binding proteins, enhancing their detoxication, or promoting their cellular export. For example, the amount of iron or cadmium in the diet influences the expression of a divalent metal transporter in enterocytes and the expression of binding proteins ferritin and metallothionein. Another example involves the cellular response to electrophiles. Figure 3–11 illustrates many genes that code for (1) enzymes that detoxify xenobiotics, (2) enzymes that eliminate ROS, (3) proteins that detoxify heme, (4) enzymes involved in glutathione homeostasis, and (5) transporters that pump xenobiotics and their metabolites out of cells. Those interested in additional examples should consult the 7th edition of Casarett & Doull's Toxicology: The Basic Science of Poisons.

When Repair Fails

Repair mechanisms often fail to protect against injury because the fidelity of the repair mechanisms is not absolute, making it possible for some lesions to be overlooked. Repair fails most typically when the damage overwhelms the repair mechanisms as when necessary enzymes or cofactors are consumed. Sometimes the toxicant-induced injury adversely affects the repair process itself. Finally, some types of toxic injuries cannot be repaired effectively, as occurs when xenobiotics are covalently bound to proteins.

It is also possible that repair contributes to toxicity, as when excessive amounts of NAD+ are cleaved by PARP when this enzyme assists in repairing broken DNA strands, or when too much NAD(P)H is consumed for the repair of oxidized proteins and endogenous reductants. Either event can compromise oxidative phosphorylation, which is also dependent on the supply of reduced cofactors (see Figure 3–8), thus causing or aggravating ATP depletion that contributes to cell injury. However, repair also may play an active role in toxicity. This is observed after chronic tissue injury, when the repair process goes astray and leads to uncontrolled proliferation instead of tissue remodeling. Such proliferation of cells may yield neoplasia, whereas overproduction of extracellular matrix results in fibrosis.

Toxicity Resulting from Dysrepair

Dysrepair occurs at the molecular, cellular, and tissue levels. Some toxicities involve dysrepair at an isolated level, such as a specific enzyme or process, or at different levels, such as tissue necrosis, fibrosis, and chemical carcinogenesis.

Tissue Necrosis

Several mechanisms that may lead to cell death may involve molecular damage that is potentially reversible by repair mechanisms. Cell injury progresses toward cell necrosis if molecular repair mechanisms are ineficient or the molecular damage is not readily reversible.

Progression of cell injury to tissue necrosis can be intercepted by two repair mechanisms working in concert: apoptosis and cell proliferation. Injured cells can initiate apoptosis, which counteracts the progression of the toxic injury by preventing necrosis of injured cells and the consequent inflammatory response.

Another important repair process that can halt the propagation of toxic injury is proliferation of cells adjacent to the injured cells. Initiated soon after cellular injury, this early cell division is thought to be instrumental in the rapid and complete restoration of the injured tissue and the prevention of necrosis. The sensitivity of a tissue to injury and the capacity of the tissue for repair are apparently two independent variables, both influencing whether tissue restitution ensues with survival or tissue necrosis occurs with death.

The efficiency of repair is also an important determinant of the dose–response relationship for toxicants that cause tissue necrosis. Tissue necrosis is caused by a certain dose of a toxicant not only because that dose ensures sufficient concentration of the ultimate toxicant at the target site to initiate injury, but also because that quantity of toxicant causes a degree of damage sufficient to compromise repair, allowing for progression of the injury. Tissue necrosis occurs because the injury overwhelms and disables the repair mechanisms, including (1) repair of damaged molecules, (2) elimination of damaged cells by apoptosis, and (3) replacement of lost cells by cell division.

Fibrosis

Fibrosis, a pathologic condition characterized by excessive deposition of an extracellular matrix of abnormal composition, is a specific manifestation of dysrepair of the injured tissue. As discussed above, cellular injury initiates a surge in cellular proliferation and extracellular matrix production, which normally ceases when the injured tissue is remodeled. If increased production of extracellular matrix is not halted, fibrosis develops.

TGF-β appears to be a major mediator of fibrogenesis. The increased expression of TGF-β is a common response mediating regeneration of the extracellular matrix after an acute injury. Normally, TGF-β production ceases when repair is complete. Failure to halt TGF-β overproduction, which leads to fibrosis, could be caused by continuous injury or a defect in the regulation of TGF-β.

The fibrotic action of TGF-β is due to (1) stimulation of the synthesis of individual matrix components by specific target cells and (2) inhibition of matrix degradation. Interestingly, TGF-β induces transcription of its own gene in target cells, suggesting that the TGF-β produced by these cells can amplify in an autocrine manner the production of the extracellular matrix. This positive feedback may facilitate fibrogenesis.

Fibrosis involves not only excessive accumulation of the extracellular matrix, but also changes in its composition. Basement membrane components, such as collagens and laminin, increase disproportionately during fibrogenesis.

Carcinogenesis

Chemical carcinogenesis involves inappropriate function of various repair mechanisms, including (1) failure of DNA repair, (2) failure of apoptosis, and (3) failure to terminate cell proliferation.

Failure of DNA Repair: Mutation, the Initiating Event in Carcinogenesis

Chemical and physical insults may induce neoplastic transformation of cells by genotoxic and nongeno-toxic mechanisms. Chemicals that react with DNA may cause damage such as adduct formation, oxidative alteration, and strand breakage. If these lesions are not repaired or injured cells are not eliminated, a lesion in the parental DNA strand may induce a heritable alteration, or mutation, in the daughter strand during replication. The most unfortunate scenario for the organism occurs when the altered genes express mutant proteins that reprogram cells for multiplication. When such cells undergo mitosis, their descendants also have a similar propensity for proliferation. Moreover, because enhanced cell division increases the likelihood of mutations, these cells eventually acquire additional mutations that may further increase their growth advantage over their normal counterparts. The final outcome of this process is a tumor consisting of transformed, rapidly proliferating cells.

Mutation of Proto-oncogenes: Proto-oncogenes are highly conserved genes encoding proteins that stimulate progression of cells through the cell cycle. The products of proto-oncogenes include (1) growth factors; (2) growth factor receptors; (3) intracellular signal transducers such as G proteins, protein kinases, cyclins, and cyclin-dependent protein kinases; and (4) nuclear TFs. Transient increases in the production or activity of proto-oncogene proteins are required for regulated growth, as during embryogenesis, tissue regeneration, and stimulation of cells by growth factors or hormones. In contrast, permanent activation and/or overexpression of these proteins favors neoplastic transformation. One mechanism whereby genotoxic carcinogens induce neoplastic cell transformation is by producing an activating mutation of a proto-oncogene. The altered gene (called an oncogene) encodes a permanently active protein that forces the cell into the division cycle.

An example of mutational activation of an oncogene protein is that of the Ras proteins. Ras proteins are localized on the inner surface of the plasma membrane and function as crucial mediators in responses initiated by growth factors (see Figure 3–7). Ras serves as a molecular switch, being active in the GTP-bound form and inactive in the GDP-bound form. Some mutations of the Ras gene dramatically lower the GTPase activity of the protein, which in turn locks Ras in the permanently active GTP-bound form. Continual rather than signal-dependent activation of Ras can lead eventually to uncontrolled proliferation and transformation.

Mutation of Tumor-suppressor Genes: Tumor-suppressor genes encode proteins that inhibit the progression of cells in the division cycle, which include cyclin-dependent protein kinase inhibitors, TFs that transactivate genes encoding cyclin-dependent protein kinase inhibitors, and proteins that block TFs involved in DNA synthesis and cell division (Figure 3–12).

The p53 tumor-suppressor gene encodes a 53-kDa protein with multiple functions. Acting as a TF, the p53 protein transactivates genes whose products arrest the cell cycle or promote apoptosis, and represses genes that encode antiapoptotic proteins. DNA damage and illegitimate expression of oncogenes stabilizes the p53 protein, causing its accumulation, inducing cell cycle arrest (permitting DNA repair) or even apoptosis of the affected cells.

Cooperation of Proto-oncogenes and Tumor-suppressor Genes in Carcinogenesis: The accumulation of genetic damage in the form of (1) mutant proto-oncogenes (which encode activated proteins) and (2) mutant tumor-suppressor genes (which encode inactivated proteins) is the main driving force in the transformation of normal cells with controlled proliferative activity to malignant cells with uncontrolled proliferative activity. Because the number of cells in a tissue is regulated by a balance between mitosis and apoptosis, uncontrolled proliferation results from perturbation of this balance.

Failure of Apoptosis: Promotion of Mutation and Clonal Growth

Preneoplastic cells, or cells with mutations, have much higher apoptotic activity than do normal cells. Therefore, apoptosis counteracts clonal expansion of the initiated cells and tumor cells. Facilitation of apoptosis can induce tumor regression, whereas inhibition of apoptosis is detrimental because mutations and clonal expansion of preneoplastic cells are facilitated.

Failure to Terminate Proliferation: Promotion of Mutation, Proto-Oncogene Expression, and Clonal Growth

Enhanced mitotic activity promotes carcinogenesis for a number of reasons:

1. Enhanced mitotic activity increases the probability of mutations. With activation of the cell-division cycle, a substantial shortening of the G1 phase occurs, and less time is available for the repair of injured DNA before replication.

2. Overproduced proto-oncogene proteins may cooperate with oncogene proteins to facilitate the neoplastic transformation of cells, thereby allowing less time for DNA methylation, which decreases gene transcription by inhibiting the interaction of TFs with the promoter region. Nonexpressed genes are fully methylated.

3. Cell-to-cell communication through gap junctions and intercellular adhesion through cadherins are temporarily disrupted during proliferation, which contributes to the invasiveness of tumor cells.

Nongenotoxic Carcinogens: Promoters of Mitosis and Inhibitors of Apoptosis

Many chemicals do not alter DNA or induce mutations yet induce cancer after chronic administration. Designated nongenotoxic or epigenetic carcinogens, these chemicals cause cancer by promoting carcinogenesis initiated by genotoxic agents or spontaneous DNA damage. Spontaneous DNA damage commonly occurs in normal human cells at a rate of 1 out of 108 to 1010 base pairs. Nongenotoxic carcinogens increase the frequency of spontaneous mutations through a mitogenic effect and by inhibiting apoptosis, thereby increasing the number of cells with DNA damage and mutations.

Cell injury evokes the release of mitogenic growth factors such as HGF and TGF-α from tissue macrophages and endothelial cells. Thus, cells in chronically injured tissues are exposed continuously to endogenous mitogens. Although these growth factors are instrumental in tissue repair after acute cell injury, their continuous presence is potentially harmful because they may ultimately transform the affected cells into neoplastic cells. It is important to realize that even epigenetic carcinogens can exert a genotoxic effect, although indirectly.

Selective or altered toxicity may be due to different or altered (1) exposure; (2) delivery, thus resulting in a different concentration of the ultimate toxicant at the target site; (3) target molecules; (4) biochemical processes triggered by the reaction of the chemical with the target molecules; (5) repair at the molecular, cellular, or tissue level; or (6) mechanisms such as circulatory and thermoregulatory reflexes by which the affected organism can adapt to some of the toxic effects. Although a simplified scheme outlines the development of toxicity (Figure 3–1), the route to toxicity can be considerably more diverse and complicated. An organism has mechanisms that (1) counteract the delivery of toxicants, such as detoxication; (2) reverse the toxic injury, such as repair mechanisms; and (3) of set some dysfunctions, such as adaptive responses. Thus, toxicity is not an inevitable consequence of toxicant exposure because it may be prevented, reversed, or compensated for by such mechanisms. Toxicity develops if the toxicant exhausts or impairs the protective mechanisms and/or overrides the adaptability of biological systems.