Lecture 6: Mechanisms of Toxicity

Question 1

Mechanisms of Toxicity

Answer 1

• Delivery: Site of Exposure to the Target
• Reaction of the Ultimate Toxicant with the Target Molecule
• Cellular Dysfunction and Resultant Toxicity
• Repair or Dysrepair

Question 2

Chemical Factors that Cause Inflammation and then Cellular Dysfunction

Answer 2

• Chemicals that cause DNA adducts
• Chemicals that cause protein adducts
• Chemicals that cause oxidative stress
• Chemicals that specifically interact with protein targets

Question 3

Chemicals that cause DNA adducts

Answer 3

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)

Question 4

Chemicals that cause protein adducts

Answer 4

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)

Question 5

Chemicals that cause oxidative stress

Answer 5

can oxidize DNA or proteins leading to DNA mutations or protein dysfunction and all of the above

Question 6

Chemicals that specifically interact with protein targets

Answer 6

• 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)

Question 7

Necrosis: unprogrammed cell death (dangerous)

Answer 7

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)].

Question 8

Apoptosis: one of the main forms of programmed cell death (not as dangerous to organism as necrosis).

Answer 8

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.

Question 9

Mechanisms of Apoptosis

Answer 9

• 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

Question 10

Phenotypes of apoptosis:

Answer 10

• Overall shrinkage in volume of the cell and its nucleus
• Loss of adhesion to neighboring cells
• Formation of blebs on the cell surface
• DNA fragmentation: dissection of the chromatin into small fragments
• Rapid engulfment of the dying cell by phagocytosis

Question 11

Factors that induce apoptosis:

Answer 11

• Internal stimuli: abnormalities in DNA

- External stimuli: removal of growth factors, addition of cytokines (tumor necrosis factor—TNF)

Question 12

Signal transduction pathways leading to apoptosis:

Two major pathways:

Answer 12

• Intrinsic pathway (mitochondria-dependent)

- Extrinsic pathway (mitochondria-independent)

Question 13

Extrinsic Apoptosis

Answer 13

• 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

Question 14

Two major classes of caspases:

Answer 14

• Initiator caspases 8,9,10: initiates the onset of apoptosis by activating the executioner caspases
• Executioner caspases 3,6,7: destroy actual targets in the cell to execute apoptosis

Question 15

Caspases target:

Answer 15

• FAK (focal adhesion kinase): inactivation of FAK disrupt cell adhesion, leading to detachment of the apoptotic cell from its neighbors
• Lamins: important component of the nuclear envelope, cleavage of lamins leads to disassembly of the nuclear lamina
• Proteins required for cell structure: actin, intermediate filaments, etc–cleavage of these proteins lead to changes in cell shape and the surface blebbing
• 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
• Enzymes involved in DNA repair

Question 16

Intrinsic Apoptosis

Answer 16

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

Question 17

Process of Intrinsic apoptosis:

Answer 17

• Bax forms homo-dimers in the presence of apoptotic signals, opening a channel that translocates cytochrome c from the intermembrane space to the cytoplasm
• Bcl2 interferes with Bax function by forming a heterodimer with Bax, closing the channel and inhibiting cytochrome c translocation
• In the cytosol, cytochrome c binds to Apaf-1 to form apoptosome
• Apoptosome recruits procaspase 9 and activates it to caspase 9
• Caspase 9 activates executioner caspases 3, 6, and 7

Question 18

Mechanisms of Necrosis

Answer 18

• 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.

Question 19

Three Primary Metabolic Disorders Jeopardizing Cell Survival:

Answer 19

I. ATP depletion
II. Sustained rise in intracellular Ca2+
III. Overproduction of ROS, RNS

Question 20

I. ATP Depletion (roles if ATP)

Answer 20

-ATP plays a central role in cellular maintenance both as a chemical for biosynthesis and as the major source of energy.
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!)
-Used in biosynthetic reactions (phosphorylation and adenylation)
-Used for signal transduction regulation (e.g. phosphorylation of receptor tyrosine kinase and kinase pathways)
-Incorporated into DNA
-Muscle contraction (actin/myosin interaction) and neurotransmission
-Polymerization of cytoskeleton (actin and tubule polymerization)
-Cell division
-Maintenance of cell morphology

Question 21

Agents That Impair ATP Synthesis

Answer 21

• Inhibitors of electron transport
  
  • Cyanide inhibits cytochrome oxidase
  • Rotenone inhibits complex I—insecticide that may be an environmental cause of Parkinson’s Disease
  • Paraquat inhibits complex I—herbicide, but also causes lung hemorrhaging in humans
• Inhibitors of oxygen delivery
  
  • Ischemic agents such as ergot alkaloids, cocaine
  • Carbon monoxide—displaces oxygen from hemoglobin
• Inhibitors of ADP phosphorylation - DDT, DIM, phytochemicals
• Chemicals causing mitochondrial DNA damage - antivirals, chronic ethanol

Question 22

II. Sustained Rise of Intracellular Ca2+ (roles of Ca2+)

Answer 22

Ca2+ is involved in :
1. signal transduction regulation (i.e. PKC activation by DAG and Ca2+) and exocytosis

2. muscle contraction (actin/myosin interaction)
3. cytoskeletal polymerization (i.e. Ca2+ inhibition of actin)
4. neurotransmission (via glutamate receptor Ca2+ channel and 	voltage-gated Ca2+ channels) and synaptic plasticity
5. enzyme induction (i.e. citrate and alpha-ketoglutarate dehydrogenases from the TCA cycle)
6. Transporters (Ca2+/ATPase, Na/Ca2+ exchanger, etc.)

Question 23

Intracellular Ca2+ levels are highly regulated

Answer 23

–The 10,000-fold difference between extracellular and cytosolic Ca2+
concentration is maintained by: impermeability of plasma membrane
to Ca2+ and by transport mechanisms that remove Ca2+ from cytoplasm
(0.1 uM inside versus 1000 uM outside).

• Ca2+ sources are from outside cell or Ca2+ stores in ER or
  mitochondria (as calcium phosphate).

Question 24

Four mechanisms of calcium elimination

Answer 24

1. Extracellular Ca2+ ATPase
2. Endoplasmic reticulum Ca2+ ATPase
3. Extracellular Na+/Ca2+ exchanger
4. Mitochondrial Ca2+ uniporter

Question 25

Excitotoxicity: Consequence of Increased Intracellular Ca2+

Answer 25

• Depletion of energy reserves—decreased mitochondrial ATP production and increased loss of ATP by activation of Ca+2-ATPase.
• Dysfunction of microfilaments—impaired cell motility, disruption in cell morphology, cellular functions
• Activation of hydrolytic enzymes—disintegration of membranes, proteins, DNA, etc.
• Generation of ROS/RNS—disintegration of membranes, proteins, DNA, etc.

Question 26

III. Oxidative Stress

Answer 26

Oxidative stress: imbalance of cellular oxidants and antioxidants in favor of oxidants.

Question 27

A. Direct generation of ROS/RNS

Answer 27

a. Xenobiotic bioactivation (i.e. carbon tetrachloride, benzene)
b. Redox cycling (paraquat, MPP+)
c. Transition metals (Fe2+, Cu2+)
d. Inhibition of mitochondrial electron transport (many phytochemicals)

Question 28

B. Indirect generation of ROS/RNS

Answer 28

a. Increased Ca2+ can cause ROS/RNS
i. Activates dehydrogenases in citric acid cycle and increases electron output (NADH and FADH2)–>leads to an increase in O2.- (superoxide) by the e- transport chain.
ii. Ca2+ -activated proteases proteolytically convert xanthine dehydrogenase to xanthine oxidase, the by-products of which are O2-. and H2O2.
iii. Neurons and endothelial cells constitutively express NOS that is activated by Ca2+ increase .NO production which reacts with O2.- to produce highly reactive ONOO- (peroxynitrite).
b. Induction of CYPs (i.e. TCDD binding AhR)

Question 29

Consequences of ROS/RNS

Answer 29

1. ROS can directly oxidize and affect protein function and can mutate DNA leading to cellular dysfunction
2. ROS/RNS oxidatively inactivate Ca2+ /ATPases and elevate Ca2+
3. ROS and RNS also drain ATP reserves:
   a. NO. is a reversible inhibitor of cytochrome oxidase
   b. ONOO- irreversibly inactivates complexes I/II/III and aconitase
   c. ROS can disrupt mitochondrial membranes and dissipate the electrochemical gradient needed for ATP synthase.
4. ONOO- induces DNA single-strand breaks, which activates poly(ADP-ribose) polymerase (PARP)—PARP transfers ADP-ribose moieties from NAD+ to PARP; consumption of NAD+ compromises ATP synthesis
5. Lipid peroxidation, cell swelling, and cell rupture

Question 30

Lipid Peroxidation

Answer 30

• Free radicals can initiate peroxidative degradation of lipids by hydrogen abstraction from fatty acids.
• The lipid radical (L.) formed is converted to the lipid peroxyl radical (LOO.) by oxygen fixation
• lipid hydroperoxide (LOOH) is then formed by hydrogen abstraction from another lipid
• lipid alkoxyl radical (LO.) is formed by the Fe(II)-catalyzed Fenton reaction
• Fragmentation leads to reactive aldehydes, including the lipid aldehyde and free radicals

Lipid peroxidation is auto-catalytic

Question 31

Organophosphate (OP) Nerve Agents

Answer 31

Organophosphorus (OP) chemical warfare agents inhibit acetylcholinesterase (AChE)

Question 32

OP Poisoning

Answer 32

Stimulating nicotinic acetylcholine receptors (nAChR) that let’s in Na+ and depolarizes the membrane, leads to opening of voltage-gated Ca2+ channels further depolarizes the membrane, letting Ca2+ in

If the person doesn’t die immediately from OP poisoning, the increased Ca2+ influx can lead to activation of apoptosis or necrosis, depletion of ATP (through overusage of Ca2+/ATPases that try to get rid of Ca2+)

This can lead to neuronal death and neural inflammation (neuroinflammation) which can further exacerbate inflammation and neuronal cell death

Question 33

Oxidized Protein Repair

Answer 33

• Protein disulfides (Prot-SS, Prot1-SS-Prot2), protein sulfenic acids (Prot-SOH) and protein methionine sulfoxides (Prot-Met=O) are reduced by thioredoxin (TR-[SH]2) or methionine sulfoxide reductase; thioredoxin is regenerated by thioredoxin reductase
• Protein glutathione mixed disulfides (Prot-SSF) are reduced by glutaredoxin; glutaredoxin is regenerated by glutathione reductas

Question 34

Peroxidized Lipid Repair

Answer 34

• Phospholipid peroxyl radicals (PL-OO.) formed from lipid peroxidation may abstract hydrogens from alpha-tocopherol (TOC-OH), which can be regenerated by glutaredoxin (GRO), which in-turn can be regenerated by glutathione reductase (GR)
• A phospholipase can cleave the fatty acid peroxide (FA-OOH), which can be reduced by glutathione peroxidase (GPX) to give FA-OH; GPX is regenerated by glutathione reductase

Question 35

Quenching of Oxidative Stress

Answer 35

Detoxification of superoxide anion radical occurs by superoxide dismutase (SOD), followed by glutathione peroxidase (GPO), and catalase (CAT).

Question 36

Chronic Non-Resolving Inflammatio

Answer 36

• While inflammation is meant as a defense mechanism against noxious insult, chronic and nonresolving inflammation can cause toxicity and many diseases.
• Tissue fibrosis also occurs from chronic inflammation, e.g. liver fibrosis, lung fibrosis, which can lead to cancer
• Chronic chemical exposures that cause cell death or oxidative stress can lead to nonresolving inflammation

Question 37

Process of Acute Inflammation

Answer 37

• Inflammatory pathway consists of inducers, sensors, mediators, and target tissues.
• Inducers initiate the inflammatory response and are detected by sensors.
• Sensors, like toll-like receptors (TLRs) are expressed on specialized sentinel cells such as macrophages, dendritic cells, and mast cells
  
  • TLRs recognize molecules broadly shared across pathogens (e.g. lipopolysaccharides, double-stranded RNA from viruses, bacterial flagella)
  • TLRs also recognize endogenous molecules associated with cell stress (e.g. fibrinogen involved in blood clotting), ATP, heat shock proteins (HSPs), HMBG1 involved in organizing DNA in the nucleosome, and self DNA

Question 38

Process of Acute Inflammation (part 2)

Answer 38

• When activated, these sensing cells secrete inflammatory mediators including cytokines (e.g. tumor necrosis factor-alpha (TNFa), interleukin-1-beta (IL-1b), and IL-6), chemokines (e.g. CCL2, CXCL8), bioactive amines (e.g. histamine), bradykinin, inflammatory lipids (eicosanoids)
• These inflammatory mediators dilate blood vessels, recruit more immune cells, and act on target tissues to eliminate the inflammatory agent, repair the tissue, and elicit changes in their functional states that optimizes their response to noxious conditions

Question 39

TNF Signaling and Effects

Answer 39

TNF binds to TNF receptors, causing the receptor to form a trimer that recruits TRADD, and can activate 3 pathways:
-Activation of NF-kB
-Activation of MAPK pathways
-Induction of death signaling
Whether a cell undergoes proliferation/inflammation or cell death depends on overall inflammatory environment (other cytokines or ROS).

Question 40

Activation of NF-kB

Answer 40

TRADD recruits TRAF2 and RIP, TRAF2 recruits protein kinase IKK, which is then activated by RIP. IKK phosphorylates IkBa, which releases NFkB to translocate to the nucleus to act as a transcriptional activator of genes involved in cell survival, proliferation, inflammation, and anti-apoptotic factors

Question 41

Activation of MAPK pathways

Answer 41

: TNF induces activation of p38-MAP kinase signaling through activation of ASK1 and MEKK1, eventually leading to the phosphorylation of MKK7 which activates JNK, which is translocated to the nucleus and activates the AP-1 transcription factor to induce cell differentiation and proliferation genes

Question 42

Induction of death signaling:

Answer 42

TNF can also induce cell death through TRADD recruiting FAS-associated protein with death domain (FADD), which recruits caspase 8, a protease that activates caspase 3, leading to apoptosis

Question 43

TNF stimulation leads to:

Answer 43

• Fever
• Chemoattractant for neutrophils
• Stimulates macrophage activation and phagocytosis
• Production of oxidative stress
• Production of other inflammatory mediators like eicosanoids
• Causes insulin resistance

Question 44

Acute Inflammation Produces ROS and RNS to Eliminate Noxious Insult

Answer 44

-Macrophages and some leukocytes recruited to the site of injury undergo a respiratory burst, producing free radicals and enzymes to destroy cellular debris and foreign particles.
1) NAD(P)H oxidase is activated in macrophages and granulocytes and produces O2.- from molecular oxygen
NAD(P)H + 2O2 –> NAD(P)+ + H+ + 2O2.-
(O2.- –> .OH via SOD and the Fenton Reaction)
2) NOS is activated in macrophages but not granulocytes by IL-1 and TNF-α
L-arginine + O2–>L-citrulline + .NO
(.NO with O2.- produces ONOO- –>.NO2 + CO3.-)
3) Myeloperoxidase is discharged by the lysosome into engulfed extracellular spaces, the phagocytic vacuoles
HOOH + H+ + Cl—>HOH + HOCl (hypochloric acid)
HOCl + O2.- –>O2 + Cl- + HO•

All these ROS/RNS are destructive products of inflammatory cells.

Although these chemicals exhibit antimicrobial activity, they can damage the adjacent healthy tissues propagating tissue injury. Thus, chronic inflammation leads to increased tissue damage.

Question 45

Process of Acute Inflammation (part 3)

Answer 45

Collectively, these inflammatory mediators act to eliminate the inflammatory agent, repair the tissue, and elicit changes in their functional states that optimizes their response to noxious conditions:

• dilate blood vessels
• recruit more immune cells
• Destroy noxious agent
• Undergo an epithelial-to-mesenchymal transition (EMT) to make the basement membrane leakier so immune cells can intravasate into tissues to the site of damage.
• Secrete growth factors to stimulate cell proliferation to repair damaged tissue
• After tissue is repaired and noxious agent is gone, inflammatory response is resolved.

Question 46

Chronic Non-Resolving Inflammation*

Answer 46

Process of Tissue Damage from Non-Resolving Inflammation caused by chronic exposure to toxicant

• Toxicant causes cellular necrosis
• intracellular contents (e.g. ATP, dsDNA, etc) activated TLRs on resident macrophages
• Macrophage activation leads to secretion of inflammatory cytokines, chemokines, eicosanoids that leads to EMT and leaky basement membrane, vasodilation, recruitment of immune cells, secretion of growth factors
• Toxicant continues to cause cell death so macrophages continue to get activated and recruited to site of injury
• Macrophages also secrete TGF-beta, TNF, platelet-derived growth factor (PDGF, insulin growth factor (IGF-1) which stimulates fibroblast proliferation and differentiation leading to excessive formation of an extracellular matrix leading to fibrosis
• Activated macrophages under respiratory burst and heightened ROS undergo necrosis further exacerbating inflammatory response, fibrosis, cell death, and tissue injury
• ROS leads to further mutations, activation of cell growth pathways, leading to cancer
• ROS, macrophages, and cancer cells along with extracellular matrix form a microenvironment that facilitates invasion, angiogenesis, and metastasis

Question 47

Inflammation and Cancer

Answer 47

• Inflammation acts at all stages of tumorigenesis
• It may contribute to tumor initiation through mutations, genomic instability
• Inflammation activates tissue repair responses, induces proliferation of premalignant cells, and enhances their survival
• Inflammation also stimulates angiogenesis, causes localized immunosuppression, and promotes the formation hospitable microenvironment in which premalignant cells can survive, expand, and accumulate additional mutations
• Inflammation also promotes metastatic spread.

Question 48

Examples of Environmental “Inflammogens

Answer 48

Stress
Bacterial/viral infections
Obesity and diabetes
Fatty foods
Pesticides
Metals
Gluten
Trichloroethylene (cleaners)
Carbon tetrachloride (cleaners, refrigerant)
Cigarette smoke
Diesel exhaust
Physical injury
Alcohol
Radiation
irritants