4.2 Apoptosis and necrosis

Cell death is a crucial process in biology, with two main types: apoptosis and necrosis. Apoptosis is programmed cell death, vital for development and homeostasis. Necrosis is accidental cell death, often caused by external factors or severe damage.

Understanding these processes is key for toxicologists. Apoptosis and necrosis have distinct morphological and biochemical differences. Various toxicants can induce either type of cell death, impacting cellular health and overall organism well-being in different ways.

Apoptosis vs necrosis

• Apoptosis and necrosis are two distinct forms of cell death that play crucial roles in tissue homeostasis, development, and disease
• Understanding the differences between these processes is essential for toxicologists to assess the impact of various toxicants on cellular health and overall organismal well-being

Programmed cell death

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• Apoptosis is a highly regulated and genetically controlled process of cell death
• Occurs as part of normal development and maintenance of tissue homeostasis
• Eliminates damaged, infected, or superfluous cells without causing inflammation or damage to surrounding tissues
• Examples include removal of interdigital webbing during embryonic development and elimination of self-reactive immune cells

Accidental cell death

• Necrosis is an uncontrolled and accidental form of cell death resulting from external factors or severe cellular damage
• Occurs in response to extreme physiological conditions, such as hypoxia, toxin exposure, or physical trauma
• Characterized by a loss of membrane integrity, cellular swelling, and release of intracellular contents into the extracellular space
• Examples include cell death due to ischemia-reperfusion injury or exposure to potent toxins

Morphological differences

• Apoptotic cells exhibit distinct morphological changes, such as cell shrinkage, chromatin condensation, and formation of apoptotic bodies
• Plasma membrane remains intact during apoptosis, preventing the release of intracellular contents and inflammation
• Necrotic cells display swelling of the cytoplasm and organelles, rupture of the plasma membrane, and spillage of cellular contents into the extracellular space
• Necrosis often leads to inflammation and damage to adjacent cells and tissues

Biochemical differences

• Apoptosis is driven by the activation of caspases, a family of cysteine proteases that cleave cellular proteins and orchestrate the cell death process
• Apoptotic cells maintain ATP levels and require energy for the execution of the cell death program
• Necrosis is characterized by a rapid depletion of ATP, leading to a failure of ion pumps and a loss of membrane potential
• Necrotic cells release damage-associated molecular patterns (DAMPs) that trigger an inflammatory response and activate the immune system

Mechanisms of apoptosis

• Apoptosis can be initiated through two main pathways: the intrinsic (mitochondrial) pathway and the extrinsic (death receptor) pathway
• Both pathways converge on the activation of caspases, which are responsible for the execution of the apoptotic process

Intrinsic pathway

• Triggered by intracellular stress signals, such as DNA damage, oxidative stress, or endoplasmic reticulum stress
• Involves the permeabilization of the mitochondrial outer membrane and the release of cytochrome c into the cytosol
• Cytochrome c binds to the adaptor protein Apaf-1, forming the apoptosome complex, which activates initiator caspase-9
• Caspase-9 then activates downstream effector caspases (caspase-3, -6, and -7), leading to the cleavage of cellular substrates and the execution of apoptosis

Extrinsic pathway

• Initiated by the binding of extracellular death ligands (Fas ligand, TNF-α) to their respective death receptors (Fas, TNFR1) on the cell surface
• Ligand-receptor interaction leads to the formation of the death-inducing signaling complex (DISC), which recruits and activates initiator caspase-8
• Caspase-8 directly activates effector caspases (caspase-3, -6, and -7) or cleaves the BH3-only protein Bid, which engages the intrinsic pathway by promoting mitochondrial outer membrane permeabilization

Caspase activation

• Caspases are synthesized as inactive zymogens and require proteolytic cleavage for activation
• Initiator caspases (caspase-8, -9) are activated by dimerization and autoprocessing upon recruitment to signaling complexes (DISC, apoptosome)
• Effector caspases (caspase-3, -6, -7) are activated by cleavage by initiator caspases and carry out the execution phase of apoptosis
• Caspases cleave a wide range of cellular substrates, including structural proteins, DNA repair enzymes, and cell cycle regulators, leading to the dismantling of the cell

DNA fragmentation

• One of the hallmarks of apoptosis is the internucleosomal cleavage of genomic DNA by caspase-activated DNase (CAD)
• CAD is normally inhibited by its inhibitor, ICAD, but during apoptosis, caspases cleave ICAD, releasing active CAD
• CAD cleaves DNA between nucleosomes, generating fragments of approximately 180 base pairs and multiples thereof
• DNA fragmentation contributes to the characteristic morphological changes observed in apoptotic cells, such as chromatin condensation and nuclear fragmentation

Mechanisms of necrosis

• Necrosis is a form of cell death characterized by a loss of membrane integrity, cellular swelling, and the release of intracellular contents, leading to an inflammatory response
• Various factors can trigger necrosis, including ischemia, toxins, and physical damage

Loss of membrane integrity

• Necrotic cells exhibit a rapid loss of plasma membrane integrity due to the failure of ion pumps and the disruption of membrane structure
• The loss of membrane integrity leads to an influx of water and ions, causing cellular swelling and eventual rupture
• The release of intracellular contents, including DAMPs, into the extracellular space triggers an inflammatory response and can cause damage to neighboring cells

Cellular swelling

• As a consequence of the loss of membrane integrity and ion pump failure, necrotic cells accumulate water and ions, leading to cellular swelling
• Swelling affects both the cytoplasm and organelles, particularly the mitochondria and endoplasmic reticulum
• Cellular swelling contributes to the eventual rupture of the plasma membrane and the release of intracellular contents

Organelle dysfunction

• Necrosis is associated with the dysfunction and damage of cellular organelles, particularly mitochondria
• Mitochondrial dysfunction leads to a rapid depletion of ATP, which further exacerbates the failure of ion pumps and membrane integrity
• Damage to the endoplasmic reticulum can cause the release of calcium into the cytosol, activating calcium-dependent proteases and phospholipases that contribute to cellular damage

Inflammatory response

• The release of intracellular contents, including DAMPs, during necrosis triggers an inflammatory response
• DAMPs, such as high mobility group box 1 (HMGB1) protein and ATP, activate pattern recognition receptors on immune cells, leading to the production of pro-inflammatory cytokines and chemokines
• The inflammatory response can cause damage to surrounding tissues and contribute to the pathogenesis of various diseases, such as ischemia-reperfusion injury and neurodegenerative disorders

Toxicants inducing apoptosis

• Various toxicants can induce apoptosis in cells, either as a primary mechanism of toxicity or as a consequence of cellular damage
• Understanding the role of apoptosis in toxicant-induced cell death is crucial for assessing the potential adverse effects of these substances

Chemotherapeutic agents

• Many chemotherapeutic drugs, such as cisplatin, doxorubicin, and paclitaxel, induce apoptosis in cancer cells as their primary mechanism of action
• These agents often cause DNA damage, oxidative stress, or mitotic spindle disruption, which activates apoptotic pathways
• The selectivity of chemotherapeutic agents for cancer cells is based on the increased susceptibility of rapidly dividing cells to apoptosis

Environmental pollutants

• Exposure to environmental pollutants, such as dioxins, polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs), can induce apoptosis in various cell types
• These pollutants often exert their effects through the aryl hydrocarbon receptor (AhR), which modulates the expression of genes involved in apoptosis and cell cycle regulation
• Pollutant-induced apoptosis can contribute to the development of diseases, such as cancer and immune system disorders

Heavy metals

• Heavy metals, including cadmium, lead, and mercury, can induce apoptosis in cells through various mechanisms
• These metals can cause oxidative stress, DNA damage, and mitochondrial dysfunction, which activate apoptotic pathways
• Chronic exposure to heavy metals has been linked to the development of neurodegenerative disorders, such as Parkinson's and Alzheimer's disease, in which apoptosis plays a role

Oxidative stress

• Toxicants that generate reactive oxygen species (ROS) or disrupt antioxidant defenses can induce apoptosis through oxidative stress
• ROS can damage cellular macromolecules, including DNA, proteins, and lipids, leading to the activation of apoptotic pathways
• Examples of toxicants that induce apoptosis through oxidative stress include paraquat, a herbicide, and acetaminophen, an analgesic drug that can cause liver damage at high doses

Toxicants inducing necrosis

• Various toxicants can cause cell death through necrosis, often due to their ability to disrupt cellular energy metabolism, cause physical damage, or induce severe oxidative stress
• Necrosis induced by toxicants can lead to tissue damage, inflammation, and the development of various diseases

Ischemia-reperfusion injury

• Ischemia-reperfusion injury occurs when blood flow is restored to a tissue after a period of ischemia, leading to the generation of ROS and the induction of necrosis
• The sudden influx of oxygen upon reperfusion can overwhelm the antioxidant defenses of the cells, causing oxidative damage and cell death
• Ischemia-reperfusion injury is a common cause of necrosis in organs such as the heart, brain, and kidneys, and can occur during organ transplantation or stroke

Metabolic poisons

• Toxicants that disrupt cellular energy metabolism, such as cyanide and carbon monoxide, can induce necrosis by causing a rapid depletion of ATP
• Cyanide inhibits cytochrome c oxidase, a key enzyme in the mitochondrial electron transport chain, leading to a failure of ATP production and cell death
• Carbon monoxide binds to hemoglobin, reducing the oxygen-carrying capacity of the blood and causing tissue hypoxia, which can lead to necrosis

Physical damage

• Physical damage to cells, such as that caused by burns, frostbite, or mechanical trauma, can induce necrosis
• The disruption of cell membranes and the destruction of cellular architecture lead to a loss of membrane integrity and the release of intracellular contents, triggering an inflammatory response
• Necrosis induced by physical damage is often accompanied by apoptosis in the surrounding tissues, as the release of DAMPs can activate apoptotic pathways in nearby cells

Chemical exposure

• Exposure to high concentrations of certain chemicals, such as acids, alkalis, or organic solvents, can cause necrosis through direct cellular damage
• These chemicals can disrupt cell membranes, denature proteins, and cause oxidative stress, leading to a rapid loss of cellular integrity and function
• Examples of chemicals that can induce necrosis include hydrochloric acid, sodium hydroxide, and carbon tetrachloride, a solvent that can cause liver damage

Detection methods

• Various methods are used to detect and distinguish between apoptosis and necrosis in cells and tissues
• These methods are essential for assessing the mode of cell death induced by toxicants and for understanding the mechanisms underlying their toxicity

Morphological assessment

• Light and electron microscopy can be used to assess the morphological changes associated with apoptosis and necrosis
• Apoptotic cells exhibit characteristic features, such as cell shrinkage, chromatin condensation, and the formation of apoptotic bodies
• Necrotic cells display cellular swelling, organelle dysfunction, and the rupture of the plasma membrane
• Histological staining techniques, such as hematoxylin and eosin (H&E) staining, can help visualize these morphological changes in tissue sections

DNA laddering

• Apoptosis is characterized by the internucleosomal cleavage of genomic DNA by caspase-activated DNase (CAD), resulting in the formation of DNA fragments of approximately 180 base pairs and multiples thereof
• These DNA fragments can be visualized as a characteristic "ladder" pattern when separated by agarose gel electrophoresis
• The presence of DNA laddering is a specific indicator of apoptosis, as necrosis typically results in random DNA fragmentation and a "smear" pattern on agarose gels

Caspase activity assays

• Caspase activation is a hallmark of apoptosis, and measuring caspase activity can help distinguish apoptosis from necrosis
• Fluorometric or colorimetric assays using synthetic caspase substrates can be used to quantify caspase activity in cell lysates
• Specific inhibitors can be used to confirm the involvement of particular caspases in the apoptotic process
• The absence of significant caspase activity is often indicative of necrosis

Annexin V staining

• Phosphatidylserine (PS) is a phospholipid normally confined to the inner leaflet of the plasma membrane, but during early apoptosis, PS is translocated to the outer leaflet
• Annexin V is a protein that binds specifically to PS and can be conjugated to fluorescent dyes, such as FITC, to detect apoptotic cells
• Flow cytometry or fluorescence microscopy can be used to quantify the percentage of annexin V-positive cells, which are considered apoptotic
• Propidium iodide (PI) is often used in conjunction with annexin V to distinguish between apoptotic and necrotic cells, as PI only stains cells with compromised membrane integrity (necrotic cells)

Physiological significance

• Apoptosis and necrosis play crucial roles in various physiological processes, including development, tissue homeostasis, and the immune response
• Dysregulation of these cell death pathways can contribute to the pathogenesis of numerous diseases

Tissue homeostasis

• Apoptosis is essential for maintaining tissue homeostasis by removing damaged, infected, or superfluous cells
• The balance between cell proliferation and apoptosis ensures that tissues maintain their proper size and function
• Dysregulation of apoptosis can lead to conditions such as cancer, where cells fail to undergo apoptosis and continue to proliferate, or atrophy, where excessive apoptosis leads to tissue loss

Embryonic development

• Apoptosis plays a critical role in shaping tissues and organs during embryonic development
• Programmed cell death is responsible for removing structures that are no longer needed, such as the interdigital webbing in developing limbs or the müllerian ducts in male embryos
• Apoptosis also helps to sculpt complex structures, such as the nervous system, by eliminating excess or improperly connected cells

Immune system regulation

• Apoptosis is crucial for the development and maintenance of a functional immune system
• During T cell development in the thymus, apoptosis eliminates self-reactive T cells, preventing autoimmunity
• Activated immune cells, such as T cells and neutrophils, undergo apoptosis after an immune response to prevent excessive inflammation and tissue damage
• Viruses and tumors can exploit the apoptotic machinery to evade immune surveillance, highlighting the importance of apoptosis in immune system regulation

Disease pathogenesis

• Dysregulation of apoptosis and necrosis can contribute to the development of various diseases
• Insufficient apoptosis can lead to the accumulation of damaged or mutated cells, promoting the development of cancer and autoimmune disorders
• Excessive apoptosis has been implicated in neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, where the loss of specific neuronal populations leads to cognitive and motor deficits
• Necrosis, due to its pro-inflammatory nature, can exacerbate tissue damage and contribute to the pathogenesis of conditions such as ischemia-reperfusion injury and neurodegenerative diseases

Toxicological implications

• The study of apoptosis and necrosis is essential for understanding the mechanisms of toxicity and the potential adverse effects of various toxicants
• Toxicant-induced cell death can lead to organ-specific toxicity, carcinogenesis, and the development of various diseases

Organ-specific toxicity

• Toxicants can induce apoptosis or necrosis in specific cell types or organs, leading to organ-specific toxicity
• For example, acetaminophen overdose can cause liver damage by inducing necrosis in hepatocytes, while cisplatin, a chemotherapeutic agent, can cause kidney damage by inducing apoptosis in renal tubular cells
• Understanding the cell death pathways involved in organ-specific toxicity can help in the development of targeted therapies and preventive strategies

Carcinogenesis

• Dysregulation of apoptosis is a hallmark of cancer, as cancer cells often acquire mutations that allow them to evade apoptosis and continue to proliferate
• Some toxicants, such as dioxins and PCBs, can promote carcinogenesis by disrupting ap