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.
Signal-transduction pathways from cell membrane receptors to signal-activated nuclear transcription factors that influence transcription of genes involved in cell-cycle regulation. The symbols of cell membrane receptors are numbered 1 to 8 and some of their activating ligands are indicated. Circles represent G proteins, oval symbols protein kinases, rectangles transcription factors, wavy lines genes, and diamond symbols inhibitory proteins, such as protein phosphatases (PTP and PP2A), the GTPase-activating protein GAP, and the inhibitory binding protein IκB. Arrowheads indicate stimulation or formation of second messengers (e.g., DAG, IP3, cAMP, and Ca2+), whereas blunt arrows indicate inhibition. Phosphorylation and dephosphorylation are indicated by +P and –P, respectively. Abbreviations for interfering chemicals are printed in black (As = arsenite; CALY = calyculin A; FA = fatty acids; FB1 = fumonisin B; MC-LR = microcystin-LR; OKA = okadaic acid; MMS = methylmethane sulfonate; PMA = phorbol miristate acetate; ROS = reactive oxygen species; SHR = SH-reactive chemicals, such as iodoacetamide; STAU = staurosporin).
In the center of the depicted networks is the pathway activated by growth factors, such as EGF, that acts on a tyrosine kinase receptor (#6), which uses adaptor proteins (Shc, Grb2, and SOS; not shown) to convert the inactive GDP-bound Ras to active GTP-bound form, which in turn activates the MAP-kinase phosphorylation cascade (Raf, MAPKK, and MAPK). The phosphorylated MAPK moves into the nucleus and phosphorylates transcription factors, thereby enabling them to bind to cognate sequences in the promoter regions of genes to facilitate transcription. There are numerous interconnections between the signal-transduction pathways. Some of these connections permit the use of the growth factor receptor (#6)–MAPK “highway” for other receptors (e.g., 4, 5, and 7) to send mitogenic signals. For example, receptor (#4) joins in via its G protein β/γ subunits and tyrosine kinase Src; the integrin receptor (#5), whose ligands are constituents of the extracellular matrix (ECM), possibly connects via G-protein Rho (not shown) and focal adhesion kinase (FAK); and the G-protein-coupled receptor (#7) via phospholipase C (PLC)-catalyzed formation of second messengers and activation of protein kinase C (PKC). The mitogenic stimulus relayed along the growth factor receptor (#6)–MAPK axis can be amplified by, for example, the Raf-catalyzed phosphorylation of IκB, which unleashes NF-κB from this inhibitory protein, and by the MAPK-catalyzed inhibitory phosphorylation of Smad that blocks the cell-cycle arrest signal from the TGF-β receptor (#9). Activation of protein kinases (PKC, CaMK, and MAPK) by Ca2+ can also trigger mitogenic signaling. Several xenobiotics that are indicated in the figure may dysregulate the signaling network. Some may induce cell proliferation by either activating mitogenic protein kinases (e.g., PKC) or inhibiting inactivating proteins, such as protein phosphatases (PTP and PP2A), GAP, or IκB. Others, for example, inhibitors of PKC, oppose mitosis and facilitate apoptosis.
This scheme is oversimplified and tentative in several details. Virtually all components of the signaling network (e.g., G proteins, PKCs, and MAPKs) are present in multiple, functionally different forms whose distribution may be cell specific. The pathways depicted are not equally relevant for all cells. In addition, these pathways regulating gene expression not only determine the fate of cells, but also control certain aspects of the ongoing cellular activity.
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.
Table 3–1 Agents acting on signaling systems for neurotransmitters and causing dysregulation of the momentary activity of electrically excitable cells such as neurons and muscle cells.1 ||Download (.pdf)
Table 3–1 Agents acting on signaling systems for neurotransmitters and causing dysregulation of the momentary activity of electrically excitable cells such as neurons and muscle cells.1
1. Acetylcholine nicotinic receptor
Muscle fibrillation, then paralysis
Ind: ChE inhibitors
Ind: botulinum toxin
Pb2+, general anesthetics
2. Glutamate receptor
Neuronal activation → convulsion, neuronal injury (“excitotoxicity”)
Neuronal inhibition → anesthesia
Kainate, domoate Quinolinate Quisqualate Ind: hypoxia, HCN → glutamate release
Ketamine General anesthetics
Protection against “excitotoxicity”
3. GABAA receptor
Muscimol, avermectins sedatives (barbiturates, benzodiazepines)
Neuronal inhibition → sedation, general anesthesia, coma, depression of vital centers
Neuronal activation → tremor, convulsion
General anesthetics (halothane)
4. Glycine receptor
CNS neurons, motor neurons
Inhibition of motor neurons → paralysis
Disinhibition of motor neurons → tetanic convulsion
Ind: tetanus toxin
5. Acetylcholine M2 muscarinic receptor
Ind: ChE inhibitors
Decreased heart rate and contractility
Belladonna alkaloids (e.g., atropine)
Increased heart rate
Atropine-like drugs (e.g., TCAD)
6. Opioid receptor
CNS neurons, visceral neurons
Morphine and congeners (e.g., heroin, meperidine)
Neuronal inhibition → analgesia, central respiratory depression, constipation, urine retention
Antidotal effects in opiate intoxication
7. Voltage-gated Na+ channel
Neurons, muscle cells, etc.
Neuronal activation → convulsion
Neuronal inhibition → paralysis, anesthesia
8. Voltage-gated Ca2+ channel
Neurons, muscle cell, etc.
Neuronal/muscular activation, cell injury
Neuronal inhibition → paralysis
9. Voltage/Ca2+-activated K+ channel
Neurons, smooth and skeletal muscle, cardiac muscle
Ba2+; apamin (bee venom), dendrotoxin, 20-HETE; hERG inhibitors (e.g., cisapride, terfenadine)
Neuronal/muscular activation → convulsion/spasm vasoconstriction PMV tachycardia (torsade de pointes)
Increased cardiac contractility, excitability
Increased neuronal excitability → tremor
11. Acetylcholine M3 muscarinic receptor
Smooth muscle, glands
|Ind: ChE inhibitors|
Smooth muscle spasm
Belladonna alkaloids (e.g., atropine)
Smooth muscle relaxation → intestinal paralysis, decreased salivation, decreased perspiration
Atropine-like drugs (e.g., TCAD) See above
Acetylcholine M1 muscarinic receptor
Neuronal activation → convulsion
Ind: ChE inhibitors
12. Adrenergic alpha1 receptor
Vascular smooth muscle
Vasoconstriction → ischemia, hypertension
Antidotal effects in intoxication with alpha1-receptor agonists
Ind: cocaine, tyramine amphetamine, TCAD
13. 5-HT2 receptor
Ergot alkaloids (ergotamine, ergonovine)
Vasoconstriction → ischemia, hypertension
Antidotal effects in ergot intoxication
14. Adrenergic beta1 receptor
Increased cardiac contractility and excitability
Antidotal effects in intoxication with beta1-receptor agonists
Ind: cocaine, tyramine amphetamine, TCAD
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.
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.
ATP synthesis (oxidative phosphorylation) in mitochondria. Arrows with letters A-D point to the ultimate sites of action of four categories of agents that interfere with oxidative phosphorylation (Table 3–2). For simplicity, this scheme does not indicate the outer mitochondrial membrane and that protons are extruded from the matrix space along the electron transport chain at three sites. βOX = beta-oxidation of fatty acids; e- = electron; Pi = inorganic phosphate; ANT = adenine nucleotide translocator; ATP SYN = ATP synthase (FoF1ATPase).
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.
Table 3–2 Agents impairing mitochondrial ATP synthesis.1 ||Download (.pdf)
Table 3–2 Agents impairing mitochondrial ATP synthesis.1
A. Inhibitors of hydrogen delivery to the electron transport chain acting on/as
Glycolysis (critical in neurons): hypoglycemia, iodoacetate, and NO+ at GAPDH
Gluconeogenesis (critical in renal tubular cells): coenzyme A depletors (see below)
Fatty acid oxidation (critical in cardiac muscle): hypoglycin, 4-pentenoic acid
Pyruvate dehydrogenase: arsenite, DCVC, p-benzoquinone
(a) Aconitase: fluoroacetate, ONOO–
(b) Isocitrate dehydrogenase: DCVC
(c) Succinate dehydrogenase: malonate, DCVC, PCBD-cys, 2-bromohydroquinone, cis-crotonalide fungicides
Depletors of TPP (inhibit TPP-dependent PDH and α-KGDH): ethanol
Depletors of coenzyme A: 4-(dimethylamino)phenol, p-benzoquinone
Depletors of NADH
(a) See group A.V.i in Table 3–3
(b) Activators of poly(ADP–ribose) polymerase, MNNG, hydrogen peroxide, ONOO–
B. Inhibitors of electron transport acting on/as
Inhibitors of electron transport complexes
(a) NADH–coenzyme Q reductase (complex I): rotenone, amytal, MPP+, paraquat
(b) Cytochrome Q–cytochrome c reductase (complex III): antimycin-A, myxothiazole
(c) Cytochrome oxidase (complex IV): cyanide, hydrogen sulfide, azide, formate, NO, phosphine (PH3)
(d) Multisite inhibitors: dinitroaniline and diphenylether herbicides, ONOO–
Electron acceptors: CCl4, doxorubicin, menadione, MPP+
C. Inhibitors of oxygen delivery to the electron transport chain
Chemicals causing respiratory paralysis: CNS depressants (e.g., opioids), convulsants
Chemicals impairing pulmonary gas exchange: CO2, NO2, phosgene, perfluoroisobutene
Chemicals inhibiting oxygenation of Hb: carbon monoxide, methemoglobin-forming chemicals
Chemicals causing ischemia: ergot alkaloids, cocaine
D. Inhibitors of ADP phosphorylation acting on/as
ATP synthase: oligomycin, cyhexatin, DDT, chlordecone
Adenine nucleotide translocator: atractyloside, DDT, free fatty acids, lysophospholipids
Phosphate transporter: N-ethylmaleimide, mersalyl, p-benzoquinone
Chemicals dissipating the mitochondrial membrane potential (uncouplers)
(a) Cationophores: pentachlorophenol, dinitrophenol-, benzonitrile-, thiadiazole herbicides, salicylate, amiodarone, perhexiline, valinomycin, gramicidin, calcimycin (A23187)
(b) Chemicals permeabilizing the mitochondrial inner membrane: PCBD-cys, chlordecone
E. Chemicals causing mitochondrial DNA damage and/or impaired transcription of key mitochondrial proteins
Antiviral drugs: zidovudine, zalcitabine, didanosine, fialuridine
Chloramphenicol (when overdosed)
Ethanol (when chronically consumed)
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.
Table 3–3 Agents causing sustained elevation of cytosolic Ca2+. ||Download (.pdf)
Table 3–3 Agents causing sustained elevation of cytosolic Ca2+.
A. Chemicals inducing Ca2+ influx into the cytoplasm
I. Via ligand-gated channels in neurons
|1. Glutamate receptor agonists (“excitotoxins”): glutamate, kainate, domoate|
|2. TRPV1 receptor (capsaicin receptor) agonists: capsaicin, resiniferatoxin|
II. Via voltage-gated channels: maitotoxin (?), HO
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 A2|
|3. Lipid peroxidants: carbon tetrachloride|
|4. Cytoskeletal toxins (by inducing membrane blebbing): cytochalasins, phalloidin|
V. From mitochondria
|1. Oxidants of intramitochondrial NADH: alloxan, t-BHP, NAPBQI, divicine, fatty acid hydroperoxides, menadione, MPP+|
|2. Others: phenylarsine oxide, gliotoxin, NO, ONOO–|
VI. From the endoplasmic reticulum
|1. IP3 receptor activators: γ-HCH (lindan), IP3 formed during “excitotoxicity”|
|2. Ryanodine receptor activators: δ-HCH|
B. Chemicals inhibiting Ca2+ export from the cytoplasm (inhibitors of Ca2+-ATPase in cell membrane and/or endoplasmic reticulum)
I. Covalent binders: acetaminophen, bromobenzene, CCl4, chloroform, DCE
II. Thiol oxidants: cystamine (mixed disulfide formation), diamide, t-BHP, O2-, and HOOH generators (e.g., menadione, diquat)
III. Others: vanadate, Cd2+
IV. Chemicals impairing mitochondrial ATP synthesis (see Table 3–3)
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:
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.
Intracellular hypercalcemia facilitates formation of ROS and RNS, which oxidatively inactivates the Ca2+ pump, aggravating the hypercalcemia.
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.
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).
“Decision plan” on the fate of injured cell. See the text for details. MOMP = mitochondrial outer membrane permeabilization; MPT = mitochondrial permeability transition; Puma = p53-upregulated modulator of apoptosis; RO(N)S = reactive oxygen or nitrogen species.
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.
Apoptotic pathways initiated by mitochondrial insult, nuclear DNA insult, and Fas or TNF receptor-1 stimulation. The figure is a simplified scheme of three pathways to apoptosis. (1) Mitochondrial insult (see text) ultimately opens the permeability transition pore spanning both mitochondrial membranes and/or causes release of cytochrome c (cyt c) from the mitochondria. Cyt c release is facilitated by Bax or Bid proteins and opposed by Bcl-2 protein. (2) DNA insult, especially double-strand breaks, activates p53 protein, which increases the expression of Bax (that mediates cyt c release) and the membrane receptor protein Fas. (3) Fas ligand or tumor necrosis factor binds to and activates their respective receptor, Fas and TNF1 receptor. These ligand-bound receptors and the released cyt c interact with specific adapter proteins (i.e., FADD, RAIDD, and Apaf-1) through which they proteolytically activate procaspases (PC) to active caspases (C). The latter in turn cleave and activate other proteins (e.g., the precursor of Bid, P-Bid) and PC-3, a main effector procaspase. The active effector caspase-3 activates other effector procaspases (PC-6 and PC-7). Finally, C-3, C-6, and C-7 clip specific cellular proteins, whereby apoptosis occurs. These pathways are not equally relevant in all types of cells and other pathways, such as those employing TGF-β as an extracellular signaling molecule, and ceramide as an intracellular signaling molecule, also exist. DFF = DNA fragmentation factor; FAK = focal adhesion kinase; PARP = poly(ADP-ribose) polymerase; SREBP = sterol regulatory element binding protein.
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.