Mechanisms of Toxicity NST110, Toxicology Department of Nutritional Sciences and Toxicology University of California, Berkeley Mechanisms of Toxicity 1. Delivery: Site of Exposure to the Target 2. Reaction of the Ultimate Toxicant with the Target Molecule 3. Cellular Dysfunction and Resultant Toxicity 4. Repair or Dysrepair Mechanisms of Toxicity 1. Delivery: Site of Exposure to the Target 2. Reaction of the Ultimate Toxicant with the Target Molecule 3. Cellular Dysfunction and Resultant Toxicity 4. Repair or Dysrepair Chemical Factors that Cause Cellular Dysfunction • Chemicals that cause DNA adducts can lead to DNA mutations which can activate cell death pathways; if mutations activate oncogenes or inactivate tumor suppressors, it can lead to uncontrolled cell proliferation and cancer (e.g. benzopyrene) • Chemicals that cause protein adducts can lead to protein dysfunction which can activate cell death pathways; protein adducts can also lead to autoimmunity; if protein adducts activate oncogenes or inactivate tumor suppressors, it can lead to uncontrolled cell proliferation and cancer (e.g. diclofenac glucuronidation metabolite) • Chemicals that cause oxidative stress can oxidize DNA or proteins leading to DNA mutations or protein dysfunction and all of the above. (e.g. benzene, CCl4) • Chemicals that specifically interact with protein targets • chemicals that activate or inactivate ion channels can cause widespread cellular dysfunction and cause cell death and many physiological symptoms—Na+, Ca2+, K+ levels are extremely important in neurotransmission, muscle contraction, and nearly every cellular function (e.g. tetrodotoxin closes voltage-gated Na+ channels) • Chemicals that inhibit cellular respiration—inhibitors of proteins or enzymes involved in oxygen consumption, fuel utilization, and ATP production will cause energy depletion and cell death (e.g. cyanide inhibits cytochrome c oxidase) • Chemicals that inhibit the production of cellular building blocks, e.g. nucleotides, lipids, amino acids (e.g. amanitin from Deathcap mushrooms) • Chemicals that inhibit enzymatic processes of bioactive metabolites that alter ion channels and metabolism (e.g. sarin inhibits acetylcholinesterase and elevates acetylcholine levels to active signaling pathways and ion channels) • All of the above can also cause inflammation which can lead to cellular dysfunction Cellular Dysfunction: Necrosis versus Apoptosis Two Forms of Cell Death 1. Necrosis: unprogrammed cell death (dangerous) A. Passive form of cell death induced by accidental damage of tissue and does not involve activation of any specific cellular program. B. Early loss of plasma membrane integrity and swelling of the cell body followed by bursting of cell. C. Mitochondria and various cellular processes contain substances that can be damaging to surrounding cells and are released upon bursting and cause inflammation. D. Cells necrotize in response to tissue damage [injury by chemicals and viruses, infection, cancer, inflammation, ischemia (death due to blockage of blood to tissue)]. 2. Apoptosis: one of the main forms of programmed cell death (not as dangerous to organism as necrosis). A. Active form of cell death enabling individual cells to commit suicide. B. Caspase-dependent C. Dying cells shrink and condense and then fragment, releasing small membrane-bound apoptotic bodies, which are phagocytosed by immune cells (i.e. macrophages). D. Intracellular constituents are not released where they might have deleterious effects on neighboring cells. Mechanisms of Apoptosis Apoptosis is a cell mechanism used to eliminate cells that contain mutations, are unnecessary, or dangerous to the body It is critical to normal embryonic development and to cancer prevention Mechanisms of Apoptosis Phenotypes of apoptosis: 1. Overall shrinkage in volume of the cell and its nucleus 2. Loss of adhesion to neighboring cells 3. Formation of blebs on the cell surface 4. DNA fragmentation: dissection of the chromatin into small fragments 5. Rapid engulfment of the dying cell by phagocytosis Factors that induce apoptosis: 1. Internal stimuli: abnormalities in DNA 2. External stimuli: removal of growth factors, addition of cytokines (tumor necrosis factor—TNF) Signal transduction pathways leading to apoptosis: Two major pathways: 1. Intrinsic pathway (mitochondria-dependent) 2. Extrinsic pathway (mitochondria-independent) Extrinsic Apoptosis • The death receptor pathway I activated by external cytokines and is mitochondria- independent • The ligands of the death receptors are members of the tumor necrosis factor (TNF) family of proteins, including TNF-alpha, Fas ligand (FasL), TRAIL/Apo2L, Apo3L • Binding of ligand to the death receptors results in homotrimerization of the receptors • Death receptors contain a death domain in the cytoplasmic region that is required for apoptosis signaling Extrinsic Apoptosis Trimerization of the receptor death domains allows binding and activation of FADD (Fas-associated death domain protein) and formation of death-inducing signaling complex (DISC), which recruits and activates procaspase 8 and 10 to caspase 8 and 10. Caspases are a family of cyteine-aspartyl-specific proteases that are activated at an early stage of apoptosis and are responsible for triggering most of the changes during apoptosis. Caspases are proteolytically activated and then diffuse into the cytoplasm to cleave target proteins Extrinsic Apoptosis Two major classes of caspases: 1. Initiator caspases 8,9,10: initiates the onset of apoptosis by activating the executioner caspases 2. Initiator caspases 3,6,7: destroy actual targets in the cell to execute apoptosis Caspases target: 1. FAK (focal adhesion kinase): inactivation of FAK disrupt cell adhesion, leading to detachment of the apoptotic cell from its neighbors 2. Lamins: important component of the nuclear envelope, cleavage of lamins leads to disassembly of the nuclear lamina 3. Proteins required for cell structure: actin, intermediate filaments, etc--cleavage of these proteins lead to changes in cell shape and the surface blebbing 4. Endonuclease CAD: responsible for chromosome fragmentation. CAD cuts DNA into small fragments. CAD normally binds to an inhibitor protein. Caspases cleaves the inhibitor protein to activate CAD 5. Enzymes involved in DNA repair Extrinsic Apoptosis Intrinsic Apoptosis Intrinsic apoptosis is mitochondria-dependent and is induced by DNA damage, binding of nuclear receptors by glucocorticoids, heat, radiation, nutrient deprivation, viral infection, hypoxia, and increased intracellular calcium concentration Process of Intrinsic apoptosis: 1. Bax forms homo-dimers in the presence of apoptotic signals, opening a channel that translocates cytochrome c from the intermembrane space to the cytoplasm 2. Bcl2 interferes with Bax function by forming a heterodimer with Bax, closing the channel and inhibiting cytochrome c translocation 3. In the cytosol, cytochrome c binds to Apaf-1 to form apoptosome 4. Apoptosome recruits procaspase 9 and activates it to caspase 9 5. Caspase 9 activates executioner caspases 3, 6, and 7 Summary of Apoptosis Mechanisms of Necrosis • Cells must synthesize endogenous molecules, assemble macromolecular complexes, membranes, and cell organelles, maintain intracellular environment, and produce energy for operation. • Agents that disrupt these functions (especially energy-producing function of the mitochondria and protein synthesis) will cause cell death. ATP-SYN: ATP synthase MET: mitochondrial electron transport NOS: nitric oxide synthase PARP: poly(ADP-ribose) polymerase ROS: reactive oxygen species RNS: reactive nitrogen species XO: xanthine oxidase ΔΨm: mitochondrial membrane potential Three Primary Metabolic Disorders Jeopardizing Cell Survival: I. ATP depletion II. Sustained rise in intracellular Ca2+ III. Overproduction of ROS, RNS I. ATP Depletion ATP plays a central role in cellular maintenance both as a chemical for biosynthesis and as the major source of energy. 1. ATP drives ion transporters such as Na+/K+-ATPase (plasma membrane), Ca2+ -ATPase (endoplasmic reticulum and plasma membrane) to maintain cellular ion gradients. (most important for necrosis!) 2. Used in biosynthetic reactions (phosphorylation and adenylation) 3. Used for signal transduction regulation (e.g. phosphorylation of receptor tyrosine kinase and kinase pathways) 4. Incorporated into DNA 5. Muscle contraction (actin/myosin interaction) and neurotransmission 6. Polymerization of cytoskeleton (actin and tubule polymerization) 7. Cell division 8. Maintenance of cell morphology ATP Production in the Mitochondria Direct Consequences of ATP Depletion ATP Depletion compromised ion pumps (eg Na/K ATPase and Ca2+-ATPases) Ca2+/Na+ levels rise intracellularly loss of ionic and volume and leads to opening of voltage-gated channels regulatory controls that depolarize membranes leading to further Ca2+ and Na+ influx into the cell cell swelling (water influx) (rise in intracellular Na+) cell lysis necrosis Agents That Impair ATP Synthesis 1. Inhibitors of electron transport 1. Cyanide inhibits cytochrome oxidase 2. Rotenone inhibits complex I—insecticide that may be an environmental cause of Parkinson’s Disease 3. Paraquat inhibits complex I—herbicide, but also causes lung hemorrhaging in humans 2. Inhibitors of oxygen delivery 1. Ischemic agents such as ergot alkaloids, cocaine 2. Carbon monoxide—displaces oxygen from hemoglobin 3. Inhibitors of ADP phosphorylation - DDT, DIM, phytochemicals 4. Chemicals causing mitochondrial DNA