NST 110: Advanced Toxicology
NST 110: Advanced Toxicology NST 110
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NST 110 MIDTERM 1 MATERIAL Lecture 1: Principles of Toxicology 1. Toxicology is the study of adverse effects of chemicals on living systems 2. Branches of Toxicology 1. Mechanistic—mechanisms by which chemicals cause toxic responses 2. Forensic—investigation of cause of death 3. Clinical—treatments for poisonings and injuries caused by chemicals 4. Environmental—effects of pollutants on environment 5. Food—adverse effects of processed or natural food components 6. Regulatory—assigns risk to substances of commercial importance. 3. Dose Determines the Toxicity 1. Effective dose, toxic dose, and lethal dose are all determined from Dose-Response curves 2. Inhibitory concentration (IC50) is determined from Concentration-Response curves 3.Therapeutic Index (TI) = LD50/ED50 or TD50/ED50 a. Larger ratio, greater safety b. Disadvantages: TI does not account for the slope of the dose-response curve; slopes must be similar or parallel, toxic responses must be the same 4. Margin of safety (MS) = LD1/ED99 a. Larger ratio, greater safety b. Advantage over TI: can have different slopes, but still need the same toxic responses a. NOAEL/LOAL – measured on toxicity curve b. NOEL/LOEL – measured on efficacy curve 5. For substances required for normal physiological function, the dose-response curves will be U or J-shaped Lecture 2: Absorption and Distribution 1. Biological factors that determine toxicity 1. Routes of Absorption: a. Ingestion, Inhalation, Dermal, Intravenous, Intraperitoneal, Intramuscular, Subcutaneous 2. Routes of Distribution: a. Systemic circulation, Portal circulation, Lymphatic system, Fat, Extracellular fluid, Organs 3. Routes of Excretion: a. Feces, Urine, Expired air, secretions 2. Absorption 1. Factors involved in absorbing a chemical a. Physiochemical properties i. hydrophilicity/ lipophilicity ii. charge/ ionization iii. molecular weight/volatility b. Route of exposure 2. Absorption across membranes a. Membrane is a phospholipid bilayer consisting of a phosphate glycerol backbone with 2 fatty acid molecules esterified to the glycerol backbone and a polar head group 3. Types of transport 1. Simple Diffusion of weak acids and bases/ hydrophobic molecules (high logP) a. Ionized form: low lipid solubility and does not permeate through membrane. b. Non-ionized form: lipid soluble, resulting in diffusion across the membrane. c. Acids are absorbed at acidic pHs (stomach) d. Bases are absorbed at basic pHs (intestine) 2. Facilitated Diffusion of hydrophilic and large molecules (low logP) a. saturable carrier-mediated transports (e.g. glucose transporter) 3. Active Transport a. chemicals are moved against an electrochemical gradient b. the transport system is saturable and requires the expenditure of energy 4. Freebase a. Refers to the deprotonated amine form of a compound, as opposed to its salt form b. Freebasing has been done with baking soda or ammonia (NH3) to increase absorption of cocaine via nasal inhalation. 4. Routes of Absorption: Ingestion 1. The GI tract a. Toxins in the GI tract do not produce systemic injury until absorbed, unless they have caustic properties b. Absorption can occur anywhere in the GI tract c. Initial metabolism can occur in gastric cells 2. GI Absorption a. Weak acids and bases: i. absorbed by simple diffusion in the non-ionized form ii. Molecules with carboxyl groups will be absorbed in stomach due to deprotonation of OH at pH 2 iii. Molecules with amine groups will be absorbed in intestine due to protonation of NH at pH 2 in stomach b. Polar substances: i. go to the liver via the portal vein. ii. first-pass metabolism or presystemic elimination in gastric/liver cells iii. can be excreted into the bile without entrance into the systemic circulation c. Non-polar substances: i. Ride on the “coat-tails” of lipids via micelles and follow lipid absorption to the lymphatic system (via chylomicrons) ii. by-pass first-pass metabolism 5. Routes of Absorption: Inhalation 1. Toxicants absorbed by the lungs include a. gases (carbon monoxide) b. vapors or volatile liquids (carbon tetrachloride) c. aerosols 2. Inhalation by-passes first-pass metabolism 3. Aerosols and Particles a. >5um – deposited in nasopharyngeal region or mouth (remove by blowing nose) b. 2-5um – deposited in tracheobronchiolar regions of lungs (remove by coughing) c. <1um – penetrates to alveolar sacs of lungs and is absorbed into blood or cleared through lymphatic system after being scavenged by alveolar macrophages in lung 6. Routes of Absorption: Dermal 1. Dermal Absorption Process a. All toxicants move through the stratum corneum (the upper most layer of the epidermis) by passive diffusion and enter systemic circulation b. rate of diffusion is proportional to lipid solubility and inversely proportional to MW 2. Dermal absorption by-passes first-pass metabolism 2. Factors that affect stratum corneum absorption a. hydration of the stratum greatly increases permeability of toxicants b. damage to the stratum c. solvent administration (carrier solvents and creams can aid in absorption) 7. Special routes of Absorption: 1. subcutaneous injection a. slow absorption b. by passes first pass metabolism directly into systemic circulation 2. intramuscular injection a. slow absorption b. by passes first pass metabolism directly into systemic circulation 3. intraperitoneal injection a. quick absorption due to high vascularization b. first pass metabolism 4. intravenous injection a. quick absorption b. by passes first pass metabolism directly into the systemic circulation 8. Toxicity is dependent on route of exposure 9. Distribution 1. The rate of distribution to organs or tissues is determined by: a. Physicochemical properties of the chemical b. Blood flow c. Rate of diffusion out of the capillary bed into the cells of an organ or tissue d. Affinity of a chemical for various tissues. 2. Distribution of toxicants can be highly localized, restricted or disperse depending on: a. Binding and dissolution into various storage sites (fat, liver, bone) b. Permeability through membranes c. Protein binding d. Active transport 10. Storage of Toxicants 1. Plasma proteins a. albumin: the most abundant plasma protein; protein ligand interactions occur through H bonding and Van der Waals forces i. bilirubin, a heme by product, is neurotoxic at high levels but is normally bound to albumin to make it less toxic 2. Liver and Kidney as storage depots – have the highest capacity of binding chemicals a. ligandin: cytoplasmic protein in the liver, has high affinity for organic acids b. Metallothionein: found in the kidney and liver, has high affinity for cadmium and zinc 3. Fat Storage a. many highly lipophilic toxicants are distributed and concentrated in fat (e.g. dioxin, DDT) 4. Bone Storage a. Storage of fluoride, lead, and strontium b. The mechanism of storage is through exchange of bone components for the toxicant 5. Effect of storage on toxicity a. Reduces toxicity of some substances by taking toxic substances out of sites of action. b. Increases toxicity if a) toxicity at storage site, b) loss of storage site c. Can produce chronic toxicity from prolonged exposure. 11. Blood Brain Barrier 1. Four Reasons why toxicants do not readily enter the CNS a. Tightly bound endothelial cells keep water-soluble substances out b. Multi-drug-resistant (mdr) protein transports some chemicals back into the blood. c. Capillaries are surrounded by glial cells, restricting access to lipid-soluble substances d. The protein concentration in the interstitial fluid of the CNS is much lower than in other body fluids. 2. Methyl Mercury transport across the blood brain barrier a. Methylmercury combines with cysteine, forming a structure similar to methionine b. is transported across the blood brain barrier through the methionine transporter b. Once in the brain, can cause neurotoxicity 3. Children are more susceptible to neurotoxicants because the blood brain barrier is not fully developed at birth 4. ATP Binding Cassette (ABC) Proteins a. membrane proteins that transport compounds through the membrane against a concentration gradient via ATP Lecture 3: Excretion 1. Routes of elimination 1. Kidney (urine) 2. Biliary (feces) 3. Lung (gases) 2. Urinary excretion 1. Glomerular filtration a. passive transport of large compounds through pores in the glomeruli capillaries b. important for maintaining blood osmolarity 2. Tubular Secretion a. active transport into renal tubules b. OCT: organic cation transporter, OAT: organic anion transporter, MDR/MRP: multidrug resistant transporters 3. Tubular Reabsorption a. passive transport of lipophilic substances b. active transport of OCT’s, peptide transporters (PEP), MRP’s b. high urinary pH = increased excretion of acids low urinary pH = increased excretion of bases *normal pH range of urine is slightly acidic: 6-6.5 3. Lungs/ Respiration 1. Respiratory acidosis a. Cause: hypoventilation b. Effect: increase PCO2, increase H+ (decrease pH) c. Effect on excretion i. acids excreted less efficiently ii. bases excreted more efficiently 2. Respiratory alkalosis a. Cause: hyperventilation b. Effect: decrease PCO2, decrease H+ (increase pH) c. Effect on excretion i. bases excreted less efficiently ii. acids excreted more efficiently 3. Metabolic acidosis a. Cause: increase in H+ caused by diarrhea; failure of kidney to secrete H+ b. Effect: decrease pH, decrease HCO3- c. Compensation: i. drives respiratory response, reduce PCO2 and restore HCO3-/CO2 ratio to 20/1 ii. renal excretion of H+, restoration of HCO3- 4. Metabolic alkalosis a. Cause: increase in HCO3- caused by ingestion of alkaline drugs; excessive vomiting b. Effects: increase pH, increase HCO3- c. Compensation: i. slows respiratory drive, elevate PCO2 and restore HCO3-/CO2 to 20/1 ii. renal excretion of excess HCO3- 5. Exhalation a. simple diffusion b. elimination of gases is roughly inversely proportional to the rate of their absorption (i.e. gases with slow solubility in blood are rapidly excreted) 4. Biliary Excretion 1. The most important source of fecal excretion of xenobiotics 2. Active Transporters on hepatic parenchymal cells 3. Enterohepatic circulation a. causes increased retention of xenobiotics conjugated by glucuronic acid because they are deconjugated in the intestine and reabsorbed b. example: DES i. endocrine disruptor, a synthetic nonsteroidal estrogen ii. was used in pregnant women to reduce complications, but caused vaginal cancer in daughters of women exposed iii. undergoes enterohepatic circulation and is retained in the body by sequential conjugation Lecture 4: Phase I Metabolism 1. Biotransformation 1. Changes the properties of a xenobiotic from a lipophilic form (favoring absorption) to a hydrophilic form (favoring excretion) 2. It can detoxify or bioactivate xenobiotics to more toxic forms 3. Xeniobiotic enzymes present in highest concentration in liver 4. Most Biotransformation occurs in the ER 5. Biotranformation is the only major defense against lipid soluble toxins 2. Phase I Biotransformation 1. hydrolysis, reduction and oxidation to expose or introduce a polar functional group 2. can generate reactive electrophiles to react with nucleophiles 3. Cytochrome P450 1. high catalytic versatility and wide array of xenobiotics that it metabolizes 2. CYP heme-containing enzymes found in the liver and ER 3. require NADH (proton donor) and Fe3+ (electron donor) 4. NADPH-Cytochrome P450 Reductase 1. CYP reductase transfers electrons from NADPH to CYP through redox with FAD and FMN NADPH à FAD à FMN à CYP 2. CYP reductase has two domains: - NADPH/FAD binding site - FMN binding site 3. CYP binding to CYP Reductase is mediated by: a. Localization b. Electrostatic Interactions: CYP has a positively charged region that interacts with negatively charged residues on CYP reductase. 5. CYP1A1 1. Organ: Lung/intestine 2. Substrates: polycyclic arylhydrocarbons (PAH), estradiol, prostaglandins 3. Inducers: substrates can induce expression 6. CYP1A2: 1. Organ: liver 2. Substrates: aromatic amines (e.g. caffeine) 3. Inducers: less inducible than CYP1A1; similar inducing agents 7. CYP2E1 1. Organ: liver 2. Substrates: alcohol, benzene, caffeine, Tylenol 3. Inducers: ethanol 4. Leads to hepatocellular necrosis and liver damage a. Forms an intermediary superoxide, which can generate hydroxyl radicals – NOT GOOD! 8. CYP3A4 1. Organ: Liver, small intestine 2. Substrates: aflatoxin, benzo(a)pyrene and other PAHs 3. Inducers: PCB, DDT, many drugs 4. CYP3A4 is the major CYP in human liver. 11. Other examples of CYP producing toxic metabolites 1. Nitrosamines – found in tobacco smoke and foods that use nitrate as a preservative 2. Ethyl carbamate – by product found in alcoholic beverages formed from urea and ethanol 12. Peroxidases (PHS, MOx, LOx) 1. Cooxidation: couple the reduction of hydrogen peroxide and lipid hydroperoxides to the oxidation of other substrates R-OOH + XB-H à R-OH + XB-O 2. Do not require reduced cofactors NADPH and NADH 3. Examples: a. Prostaglandin H synthase (PHS, COX) b. Myeloperoxidase (MOx) and Lactoperoxidase (LOx) 12. Benzene 1. Target: liver, kidney, lung, heart, and brain 2. Effect: DNA strand breakage, chromosomal damage, protein binding—can cause bone marrow suppression and leukemia 3. Exposure: vapors from glues, paints, Air around hazardous waste sites or gas stations 13. FMOs 1. Oxidize nucleophilic N, S, and P heteroatoms of a variety of xenobiotics 2. FMOs are not inducible and are constitutively expressed. 3. Can be inhibited by other substrates. 4. Located in microsomal fraction of liver, kidney, and lung. 15. EHs 1. Catalyzes the trans-addition of water to alkene epoxides 2. EH enzymes are found in virtually all tissues 3. EH primarily acts as a detoxification enzyme 4. EH Induction a. CYP inducers, PAH and TCCD b. antioxidants BHA and BHT c. antioxidant defense glutathione S-transferase and reductase 16. Benzo[a]pyrene (BaP) 1.Pott 1775 – soot caused scrotal cancer; what was in the soot? 2. BAP is a polycyclic aromatic hydrocarbon and a potent carcinogen upon bioactivation 3. Found in charbroiled meats, tobacco smoke, and coal tar 4. When BAP reacts with DNA, it causes a G to T mutation 17. Aflatoxins 1. naturally occurring mycotoxins that are produced by many species of Aspergillus, a fungus. 2. They can be found on moldy peanuts, rice, corn and other crops. 3. Aflatoxin B1 is the most potent liver carcinogen (in its aflatoxin-epoxide form) 18. Carboxylesterases (CES) 1. Hydrolyze a carboxyl-ester into a carboxylic acid and ester Lecture 5: Phase II Metabolism 1. Phase II Biotransformation 1. Reactions include glucuronidation, sulfation, glutathione conjugation, and conjugation with amino acids that strongly increase hydrophilicity. 2. Glucuronidation 1. Substrates: nucleophiles and carboxylic acids 2. Enzymes: UGT a. Low Affinity, High Capacity Enzyme (work better at higher doses) 3. Cofactor: UDP-GA 4. Excretion a. <350 Da excreted via kidney and urine a. >350 Da excreted via Enterohepatic Circulation (bile) i. delays the elimination of xenobiotics and can increase toxicity. 3. Sulfation 1. Substrates: nucleophiles (NOT carboxylic acids) 2. Enzymes: Sulfotransferases (SULT) a. High Affinity, Low Capacity Enzyme (work better with lower doses) 3. Cofactor: PAPS 4. Excretion a. actively excreted in the urine by organic anion transporters b. hydrolysis by arylsulfatases can contribute to enterohepatic circulation 4. Glutathionylation 1. Substrates: electrophiles 2. Enzymes: Glutathione S-transferase (GST) 3. Cofactor: GSH 4. Excretion: a. conjugates can be formed in the liver and excreted in bile b. can be converted to mercapturic acids in the kidney and excreted in the urine 5. Acetylation 1. Substrates: nucleophiles 2. Enzymes: N-Acetyltransferases (NAT) 3. Cofactor: Acetyl CoA 4. Excretion a. products are less water soluble than the parent compound b. can now undergo many oxidation reactions 6. Methylation 1. Substrates: nucleophiles 2. Enzymes: Methyltransferases (MT) 3. Cofactor: SAM 4. Excretion a. products are less water soluble b. exception: methylation of nicotine increases solubility Lecture 6: Bioactivation and Gene Regulation 1. Regulation of Xenobiotic Metabolism 1. Induction of xenobiotic metabolism enzymes occurs at a genetic level a. CYPs: PAHs, TCDD (dioxin), phenobarbital b. UGTs: co-induced with CYPs c. GST/EH/quinone reductases: PAHs, electrophiles, oxidative stress d. CAR: activation of CAR à nuclear translocation of CAR à transcription e. PXR, AhR: XB binds nuclear receptor à nuclear translocation à binding to promoter 2. Xeniobiotic Response Element (AhR-ARNT) 1. AhR: a ligand-activated transcription factor present in the cytoplasm 2. AhR exists in the cytoplasm in association with chaperones (HSP90, p23) 3. Ligand binds AhR, releasing AhR from chaperones a. Ligands: PAH, TCDD (dioxin), Bilirubin (endogenous), tryptamine (endogenous) 4. Free AhR translocates into the nucleus 5. AhR heterodimerizes with ARNT 6. AhR/ARNT binds the XRE/DRE a. CYP promoter region, activates gene transcription 7. The AhR repressor (AhRR) can displace AhR/ARNT 8. AhR is exported to cytoplasm and degraded by proteasomes 3. Antioxidant Response Element (Nrf2-KEAP) 1. Nrf2: the principal transcription factor present in the cytoplasm 2. KEAP1: repressor protein, binds to Nrf2 and retains it in the cytoplasm a. promotes proteasome degradation of Nrf2 3. Oxidation of thiol groups to dithiols on KEAP1 causes Nrf2 to be released 4. Free Nrf2 is phosphorylated and translocates into the nucleus 5. Nrf2 heterodimerizes with MAF 6. Nrf2/MAF binds to the ARE a. activates transcription of antioxidant/detoxification enzymes 7. Nrf2 can separate from MAF 8. Nrf2 is exported to cytoplasm and degraded by proteasomes Lecture 7: Mechanisms of Toxicity 1. Four Step Mechanism 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 (detoxification) or disrepair (bioactivation) e.g. if you were constantly being exposed to epoxides, you will start to deplete your GST stores which can lead to an inability to detoxify 2. Chemical factors that cause cellular dysfunction 1. Chemicals that cause DNA adducts lead to DNA mutations a. activation of oncogenes/ inactivation of tumor suppressors à cancer b. e.g. benzopyrene 2. Chemicals that cause protein adducts lead to protein dysfunction a. activation of oncogenes/inactivation of tumor suppressors à cancer b. e.g. glucoronidation metabolite 3. Chemicals that cause oxidative stress initiate DNA mutations and protein dysfunction a. e.g. benzene 3. Chemicals that specifically interact with protein targets a. inactivate ion channels (tetrodotoxin) b. inhibit cellular respiration (cyanide) c. inhibit enzymatic processes (sarin) d. inhibit production of cellular building blocks (deathcap mushrooms) 4. All of the above cause inflammation 3. Apoptosis: Programmed Cell Death 1. Active form of cell death used to eliminate unnecessary or dangerous cells 2. Caspase-dependent 3. Dying cells shrink (chromatin compaction) and then fragment, releasing small membrane- bound apoptotic bodies, which are phagocytized by macrophages 4. Intracellular constituents are not released where they might have delirious effects on neighboring cells 4. Extrinsic Approach (mitochondria-independent) 1. External stimuli: removal of growth factors, addition of cytokines (tumor necrosis factor TNF) 2. Mechanism a. The death receptor pathway is activated by external ligands i. TNFR1, Fas ligand (FasL), TRAIL/Apo2L, Apo3L b. Binding of ligand results in homotrimerization of the receptor death domain c. Causes activation of FADD and formation of DISC d. DISC recruits and activates Procaspase 8 and 10 and cleaves to Caspase 8 and 10 i. intiator caspases: initiate apoptosis by activating executioner caspases e. Caspase 8,10 activate Caspase 3, 6, 7 i. executioner caspases: destroy actual targets to execute apoptosis f. Caspase 3,6,7 target i. FAK (cell adhesion) ii. Lamins (nuclear envelope) iii. Proteins required for cell structure (actin) iv. Endonuclease CAD v. Enzymes involved in DNA repair 5. Intrinsic Approach (mitochondria-dependent) 1. Internal stimuli: abnormalities in DNA 2. Mechanism a. Death receptor pathway is activated by intrinsic apoptosis signals i. DNA damage, binding of nuclear receptors by glucocorticoids, heat, radiation, nutrient deprivation, viral infection, hypoxia, and increased intracellular [Ca2+] b. Presence of intrinsic signals results in homodimerization of Bax i. Bcl2 inhibits homodimerization of Bax c. Opens a channel allowing translocation of cytochrome c from the intermembrane space to the cytoplasm d. cytochrome c binds to Apaf-1 to form apoptosome e. Apoptosome recruits procaspase 9 and activates it to caspase 9 i. intiator caspases: initiate apoptosis by activating executioner caspases f. Caspase 9 activates Caspases 3, 6, 7 i. executioner caspases: destroy actual targets to execute apoptosis g. Caspase 3,6,7 target i. FAK (cell adhesion) ii. Lamins (nuclear envelope) iii. Proteins required for cell structure (actin) iv. Endonuclease CAD v. Enzymes involved in DNA repair 6. Necrosis: Unprogrammed Cell Death 1. Passive form of cell death induced by accidental damage of tissue and does not involve any specific cellular program 2. Early loss of plasma membrane integrity resulting in swelling and bursting of cell 3. Mechanisms of Necrosis a. ATP Depletion b. Sustained rise in Intracellular Ca2+ c. Overproduction of ROS/RNS 7. ATP Depletion a. inhibitors of electron transport i. cyanide inhibits cytochrome oxidase ii rotenone and paraquat inhibit complex I b. inhibitors of oxygen delivery i. ischemic agents like cocaine ii. carbon monoxide which displaces O2 from hemoglobin c. inhibitors of ADP phosphorylation i. DDT d. chemicals causing mitochondrial DNA damage i. antivirals, chronic ethanol 8. Sustained Rise of Intracellular Calcium 1. Ca2+ Functions a. signal transduction regulation (activation of PKC), exocytosis, muscle contraction, cytoskeletal polymerization, neurotransmission, enzyme induction, transporters 2. Regulation of Intracellular Ca2+ levels a. impermeability of the plasma membrane to Ca2+, transport mechanisms that remove Ca2+ outside of the cell, storage of Ca2+ in ER or mitochondria 3. Mechanisms of Calcium Elimination a. Extracellular Ca2+ ATPase à pump Ca2+ out of the cell into the region of higher Ca2+ concentration requires ATP and electron carriers NADPH b. ER Ca2+ ATPase c. Extracellular Na+/Ca2+ exchanger d. Mitochondrial Ca2+ uniporter 4. Excitotoxicity: Consequence of increased Ca2+ levels a. depletion of energy reserves b. dysfunction of microfilaments c. activation of hydrolytic enzymes d. generation of ROS/RNS – disintegration 9. Reactive Oxidative Stress and Reactive Nitrogen Species (ROS/RNS) 1. Direct generation of ROS/RNS a. Xenobiotic bioactivation (benzene) b. redox cycling (paraquat) c. transition metals (Fe2+, Cu2+) d. inhibition of mitochondrial electron transport (cytochrome c) 2. Indirect generation of ROS/RNS a. increased Ca2+ i. activates dehydrogenases in TCA, increasing electron output, increasing O2 ii. convert xanthine dehydrogenase to xanthine oxidase, increasing O2 iii. neurons express NOS that is activated by Ca2+ increasing NO- which reacts with O2- to produce ONOO- 3. Consequences of ROS/RNS a. can directly oxidize and affect protein function and can mutate DNA b. inactivate Ca2+ ATPases and elevate Ca2+ c. drain ATP reserves i. NO- is reversible inhibitor of cytochrome oxidase ii. ONOO- irreversibly inhibits complexes I/II/III d. ONOO- induces DNA single strand breaks e. lipid peroxidation, cell swelling, cell rupture 10. Lipid Peroxidation 1. Free radicals can initiate peroxidative degradation of lipids by dehydrogenation of fatty acids. 2. The lipid radical (L) à lipid peroxyl radical (LOO) by oxygen fixation 3. (LOO) à lipid hydroperoxide (LOOH) by hydrogen abstraction from another lipid 4. (LOOH) à lipid alkoxyl radical (LO.) by the Fe(II)-catalyzed Fenton reaction 5. Fragmentation leads to reactive aldehydes, including the lipid aldehyde and free radicals 6. Lipid peroxidation is auto-catalytic!!! 11. Example of Energy Depleting Neurotoxin: MPTP 1. MPTP: a contaminant in MPPP 2. Causes Parkinson’s Disease through selective degeneration of dopaminergic neurons in the substantia nigra 3. Mechanism a. MPTP crosses the blood brain barrier (hydrophobic) b. MPTP gets bioactivated to MPP+ by MAOB found in glial cells in the brain c. MPP+ is selectively taken up by dopamine transporters in the brain d. MPP+ inhibits complex I of the electron-transport chain and causes oxidative stress in dopaminergic neurons to cause neurodegeneration. e. patients develop irreversible symptoms of Parkinson’s disease f. MAOB inhibitors (selegiline) are used to prevent conversion of MPTP to MPP+ g. MPP+ can also undergo quinone cycling and cause oxidative stress 4. Repair Mechanisms a. Oxidized protein repair i. Protein disulfides/sulfenic acids/methionine sulfoxides reduced by thioredoxin ii. Protein glutathione mixed disulfides are reduced by glutaredoxin pathways b. Peroxidized lipid repair i. phospholipid peroxyl radicals formed from lipid peroxidation may abstract hydrogens from tocopherol, quenching the radical ii. tocopherols can then regenerate its hydrogen, quenching its radical c. DNA repair i. alkyltransferases, nucleotide excision, double strand break repair, mismatch repair 12. Chronic Non-Resolving Inflammation 1. Chronic toxic exposure à cellular necrosis à activation/recruitment of resident macrophages à Macrophages secrete proteases that degrade the ECM à fibrosis à induce cell proliferation and metastasis (promotion) by taking advantage of the leaky membrane 2. Examples: lung tissue damage from asbestos 13. Acute Inflammation 1. Inducers: initiate the inflammatory response 2. Sensors: toll like receptors are expressed on specialized cells, such as macrophages; recognize pathogens and endogenous molecules 3. Mediators: sensors secrete inflammatory mediators such as a. cytokines (TNF, IL-1, IL-6) b. chemokines (CCL2, CXCL8) c. bioactive amines (histamine) d. inflammaotry lipids (eicosanoids) 4. Target Tissues a. dilate blood vessels, recruit immune cells, stimulate EMT (increase permeability of basement membrane to immune cells), destroy noxious agent 5. Acute inflammation produces ROS and RNS to eliminate noxious insult a. NADPH oxidase: activated in macrophages and produces O2- from molecular oxygen NADPH + 2O2 à NADP+ + 2O2- + H+ b. NOS: activated in macrophages by IL-1 and TNF-α c. Myeloperoxidase: discharged by the lysosome into engulfed extracellular spaces, the phagocytic vacuoles 14. Neuroinflammation and Neurodegenerative Diseases Microglia are a type of glial cell that are the resident macrophages of the brain and spinal cord, and thus act as the first and main form of active immune defense in the central nervous system (CNS) 1. Alzheimer’s i. Amyloid-beta peptide, produced by cleavage of amyloid precursor protein (APP), forms aggregates that activate microglia 2. Parkinson’s a. loss of dopaminergic neurons in the substantia nigra of the midbrain and the presence of intracellular aggregates of alpha-synuclein protein activate microglia b. possible environmental agents: Rotenone and Paraquat – Inhibit complex I of ETC 15. Inflammation and Cancer 1. Contribute to tumor initiation through mutations 3. Activate tissue repair responses 4. Promote proliferation of premalignant cells and their survival 5. Promote metastasis 6. Stimulate angiogenesis and causes localized immunosuppression Lecture 8: Carcinogenesis 1. Process of Carcinogenesis 1. Initiation: mutation in one or more genes controlling regulatory pathways of the cell 2. Promotion: selective growth enhancement induced by exposure to a promoting agent 3. Progression: results from recruitment of immune cells that promote inflammation, mutation, etc *These do not necessarily have to occur in this order 2. Initiation 1. Mechanism of mutation a. Point mutations: the replacement of a single nucleotide base with another nucleotide b. Frame shift mutations: addition or deletion of a nucleotide c. Chromosomal aberrations: any change in the normal structure or number of chromosomes i. Aneuploidy: chromosome number is not a multiple of the normal haploid (23) ii. Polyploidy: more than twice (46) the haploid number of chromosomes 2. Failure of DNA repair mechanisms due to a. carcinogen-induced mutational inactivation of DNA repair enzymes b. failure of the DNA repair mechanisms to recognize carcinogen-induced mutation. 3. Targets of Initiation a. Mutational activation of oncogenic (proliferative) pathways b. Mutational inactivation of apoptotic (cell death) pathways c. Mutational inactivation of DNA repair mechanisms (e.g. BER, NER, etc). d. Mutational inactivation of antioxidant response (e.g. SOD). 4. Tumor Suppressor p53 a. p53: transcriptional factor that controls cell cycle, apoptosis, and DNA repair b. Mdm2 is a negative regulator of p53 i. functions both as an E3 ubiquitin ligase and an inhibitor of p53 c. Carcinogens often mutationally inactivate p53 as well as proteins that control p53 function i. too much Akt or MAP kinase, it will phosphorylate Mdm2 and release p53 d. Benzopyrene leads to mutations in K-Ras and p53 i. Transversion mutation: G is replaced by T; original G-C base pair replaced by T-A ii. p53 and K-Ras: two genes most frequently mutated in smoking-induced lung cancers 3. Promotion 1. Mechanism a. Epigenetic event: change in gene expression without change in DNA. b. Mitogenic: Stimulates proliferation of both mutated and normal cells i. Enhances the effect of the genotoxic initiating agent by establishing clones of initiated cells d. Promotion is reversible 2. Example Promoter Agents a. ROS and redox active xenobiotics and metals b. Phorbol esters (TPA) c. Polycyclic aromatic compounds (Dioxin) d. Peroxisome Proliferators (oxidized fats) e. endocrine disruptors and growth factors 3. Endocrine Disruptors a. ERβ/ERα (estrogen receptor) ratio is decreased in cancers i.ERα-ERα homodimer leads to mitogenic activation ii. ERβ-ER heterodimer leads to growth arrest iii. ERβ inhibits ERα c. Androgen Receptor (prostate) i. AR can homodimerize with ERα leading to mitogenic activation ii. AR can heterodimerize with ERβ to cause growth arrest 4. Progression 1. Mechanism a. irreversible process that leads to metastasis b. requires further mutation and recruitment of inflammatory immune cells to the tumor 2. Example Progresser Agents a. inflammation, asbestos fibers, benzene, benzoyl peroxide, oxidative stress NST 110 MIDTERM 2 MATERIAL Lecture 1: Neurochemistry 1. Basics of Neurochemistry and Neurobiology 1. Repeated outbreaks of neurologic disease a. pesticide poisonings (organophosphates) b. methyl mercury c. chemical warfare agents (sarin) d. lead 2. Chronic, low-level environmental exposure can have potential affects on brain function 3. Neurotoxins target specific vulnerable processes of the brain a. neurotransmission b. high energy requirements c. irreversible neuron degeneration 2. Types of Neurodegeneration 1. Neurotransmission Toxicity: caused by exciting or suppressing neurons a. prolonged excitation will lead to seizure and death b. prolonged suppression will lead to sedation and paralysis 2. Neuropathy: results from the death of the entire neuron 3. Axonopathy: occurs when the axon degenerates; if neuron still intact, axon can regenerate 4. Myelinopathies: disruption of myelin resulting in dissipation of neural impulses (MS) 3. Neurotransmission 1. Inside Neuron: negatively charged (less Na+) 2. Outside of Neuron: positively charged (more Na+) 3. Generation of the Action Potential a. Depolarization: Na+ channels open; drives Na+ into the cell making the inside membrane potential positive (-70mV à +30mV) b. Repolarization: K+ channels open; drives K+ out of the cell returning the cell to its negative resting potential (+30mV à -70mV) c. Restoration of Ion Gradient: Na+/K+ ATPase i. 3 Na+ out, 2 K+ in d. Wave of Depolarization: Propagation of action potential down the axon is mediated by voltage gated Na+ channels 4. Excitotoxicity and Seizures 1. Excessive Ca2+ can cause excessive muscular contractions Mode of Action 1. Ach binds to receptors on sarcolemma 2. action potential travels down T tubule 3. Ca2+ is released from sarcoplasmic reticulum 4. Ca2+ binds troponin, exposing active site on actin 5. Myosin heads form cross bridge with actin 6. power stroke: myosin bends, ADP released 7. recovery stroke: ATP attaches to myosin, causing cross bridge to detach 2. Excessive Ca2+ Can Cause Neurodegeneration a. Triangle of Death b. Ways to eliminate or sequester intracellular calcium i. ER-Ca2+ channel, Na+/Ca2+ exchanger, Ca2+ATPases Lecture 2: Neurotransmitters 1. Acetylcholine (excitatory) 1. Acetylcholine binds nicotinic and muscarinic Ach receptors 2. Nicotinic AchR on Sympathetic Neurons (ion channel) a. Ach binding to nAchR opens Na+ channels b. triggers sympathetic response: motor control and memory c. hyperstimulation: convulsions and seizures 3. Muscarinic AchR on Parasympathetic Neurons (GPCR) a. Ach binding to mAchR raises cAMP levels b. triggers parasympathetic response c. hyperstimulation: SLUDGE (salivation, lachrymation, urination, defecation, GI mobility, emesis) 4. Degenerated by acetylcholinesterase (AchE) into acetate and choline 5. Inhibitors of AchE a. Organophosphates (OP): irreversible inhibitor b. Fasciculin c. Galantamine i. used in treatment of mild to moderate Alzheimer’s d. Physostigmine i. produced by unripe eggplant, oranges, tomatoes 6. Stimulators of AchE a. 2-pralidoxime chloride (2-PAM) 7. mAchR antagonists a. Atropine 8. nAchR agonists a. Nicotine 2. Glutamate (excitatory) 1. Glutamate binds glutamate receptors a. GluR: excite neurons by transporting Ca2+ into neurons 2. Functions a. Mediates most of the excitatory neurotransmission in the CNS b. dysfunction has been associated with epilepsy, neurodegenerative/mood disorders 3. GluR agonists a. Kainate acid, Domoic acid 3. GABA (inhibitory) 1. GABA binds GABA receptors a. GABA R: depress neurons by transporting Cl- into neurons 2. Functions a. GABA is the major inhibitory neurotransmitter in the CNS 2. GABA R antagonist c. Bicuculline: causes epilepsy and seizures via neuronal excitation 4. GABA R agonists: a. Barbiturates: used alongside a paralytic for lethal injections b. Benzodiazepines: diazepam (valium), lorazepam 4. Voltage Gated Na+ Channels (excitatory) 1. Na+ channels open upon excitation caused by other excitatory neurotransmitters 2. Tetrodotoxin: binds to and permanently closes Vg Na+ channels Lecture 3: Drugs of Abuse 1. Cannabinoid Type 1 Receptor Stimulants 1. Mode of Action a. The CB1 receptor (GPCR) is on the presynapse b. ligand binding to CB1-R suppresses neurotransmission c. Immediate effects 1. inhibits adenylate cyclase à lower cAMP à lower PKA 2. activates K+ channels à hyperpolarization 3. inhibits Ca2+ channels à suppress neurotransmission 4. inhibits release of glutamate à suppress neurotransmission d. Long-term effects 1. inhibits release of glutamate à long-term impairments in memory 2. inhibits adenylate cyclase à inactivation of PKA à inactivation of CREB 2. Marijuana (Δ9-tetrahydrocannabinol) a. mild euphoric and relaxing high, increased appetite, reversible cognitive impairments b. effects begin immediately after the drug enters the brain and last for 1-3 hours 3. Endocannabinoids a. retrograde messengers: synthesized in the post-synapse, release and bind to CB1-R on presynapse to inhibit neurotransmitter release 4. Spice a. synthetic cannabinoid b. 10-1000 times more potent 5. Rimonabant: CB1 receptor antagonist a. shown to cause weight loss and improved glucose tolerance, but was counteracted by severe depression and suicidal tendencies 2. Arachidonic Acid Inhibitors 1. Mode of Action a. Arachidonic acid is converted to prostaglandins via COX 3. Shut off arachidonic acid supplying this cascade Ananadamide (AEA) à Arachidonic Acid via FAAH 2-Arachidonyl glycerol (2AG) à Arachidonic Acid via MAGL 3. Opioid Receptor Stimulants 1. Mode of Action a. μ – Opioid receptors (GPCR) b. binding to OpioidR suppresses neurotransmission c. closes Ca2+ channels, opens K+ channels à hyperpolarization of pre and postsynapse d. Inhibits adenylate cyclase and lowers cAMP levels – lowers synaptic plasticity 2. Endogenous opioids a. endorphins: produced in the brain, bind opioid receptors 3. Morphine Diacetate (heroin) a. “prodrug”: must be metabolized to morphine for its action on OpioidR 4. Psychomotor Stimulants 1. Mode of Action a. inhibit the reuptake of dopamine, serotonin, and norepinephrine b. increase the levels of these neurotransmitters at the synapse à hyperstimulation 2. Cocaine, Amphetamines, Methamphetamines, MDMA 3. Adrenergic receptor stimulation: flight or fight response Lecture 4: Drug Addiction 1. Addiction 1. Tolerance: the need for increasing doses of a drug to achieve the same effect 2. Desensitization: prolonged overstimulation; require more drug to achieve the desired effect 3. Dependence: an adapted physiological state that develops to compensate for excessive stimulation by a drug 3. Withdrawal: unmasking of the adapted state when drug intake stops 2. Dopamine Mediates Reinforcement 1. Heightened dopamine levels over long-periods of time will lead to desensitization of dopamine receptors requiring more drug to get the same level of happiness. 2. Reinforcing and dependence effects of drugs involve indirect modulation of dopamine 3. Ventral Tegmental Area (VTA): origin of the dopaminergic cell bodies 1. amphetamines inhibit reuptake of dopamine à elevate the levels of dopamine à stimulate dopamine receptors on dopamine releasing neurons à increased release of dopamine into nucleus accumbens 2. opioids, cannabinoids stimulate their receptors on GABA releasing neurons à decreases GABA release à disinhibition of dopamine neurons and increased release of dopamine into nucleus accumbens 3. nicotine stimulates nAchR on Glutamate releasing neurons à Glu binds to GluR on dopamine releasing neurons à increased release of dopamine into nucleus accumbens 4. Neuronal Circuitry of Addiction 1. reinforcing action of drugs is mediated through activation of mesocorticolimbic dopamine neurons in the VTA of the midbrain 2. elevation of dopamine in the nucleus accumbens is particularly important since it serves at the interface between the limbic and cortical regions (important for motivation and pleasure) 5. Dopaminergic System 1. Five Dopaminergic receptor types (D1-D5) 2. D1 receptor (GPCR) a. stimulates GluR and opens Ca2+ channels b. stimulates gene expression of CREB (protein involved in synaptic plasticity) and long term potential (LTP) which strengthens the connection between the substance and reward Lecture 5: Toxicants that Cause Neurodegeneration 1. The Brain’s Energy Requirements 1. Neurons are highly dependent on energy to maintain ion gradients 2. Ischemia: the interruption of blood flow (O2 and glucose) to the brain 3. Hypoxia: the lack of oxygen, can also cause major CNS damage (e.g. cyanide, carbon monoxide). 2. MPTP: Energy Depleting Neurotoxin 1. MPTP: contaminant in MPPP, an opioid analgesic drug 2. Mechanism a. MPTP crosses the blood brain barrier b. MPTP is metabolized to the bioactivated agent MPP+ by MAOB in glial cells in the brain c. MPP+ is selectively taken up by dopamine transporters in the brain d. MPP+ inhibits complex I of the electron transport chain and depletes ATP in neurons e. Develop irreversible symptoms of Parkinson’s disease f. Antidote: MAOB inhibitors such as selegiline 3. Environmental Cause of Parkinson’s 1. 95% environmental component 2. Parkinson’s symptoms not observed until 80% of substantia nigra neurons are lost 3. Rotenone: insecticide that inhibits complex I of the ETC 4. Paraquat: herbicide that produces oxidative stress similar to MPP+ 4. Neuroinflammation is a Hallmark of Neurodegenerative Disease Lecture 6: Pesticide Toxicology 1. Pesticide: substance or mixture of substances intended for preventing, repelling, or mitigating pests 2. Safety and Selective Toxicity 1. Selective toxicity is the basis for pesticide safety 2. Selectivity is a compromise between pesticidal potency and human/animal/crop safety 3. Broad spectrum/non-selective vs Narrow spectrum/highly selective Insecticides 1. Organochlorines: DDT 1. Stimulate Na+ channels 2. Bio accumulates in fat, persistent 3. Endocrine disruptor: binds and stimulates the estrogen receptor to cause aberrant cell growth 4. Potential carcinogen: can induce CYPs, cause oxidative stress 3. DDT Environmental Impacts and Ban a. Rachel Carson’s “Silent Spring” b. Environmental effects: clear lake, DDT almost wiped out American Bald Eagle c. Broad insect resistance 2. Pyrethroids 1. Stimulate Na+ channels 2. Selective for insects 3. Widely used due to fast biodegradability 3. Chlorinated Hydrocarbons 1. GABA Receptor antagonists (inhibit inhibition) 2. bio accumulative, persistent 3. carcinogenic: can cause CNS convulsions 4. Neonicotinoid 1. nAchR agonist 2. derived from nicotinoid structures 3. higher insect toxicity than mammalian – low selectivity within insects a. negative tip on neonicotinoids replaces positively charged tip on nicotine -- confer selectivity for insect nicotinic receptors 5. Organophosphates (OP) (Chlorpyrifos) 1. Irreversible inhibitor of AchE 2. Off Target effects 3. Treatment: Atropine (mAchR antagonist) or 2-PAM (AchE agonist) 6. Using Reactivity-Based Protein Profiling (RBPP) to Identify Off-Targets of OP Pesticides 1. treated proteome binds to enzyme; when visualized under probe, less probe binds; under gel, you will see a blank spot where probe wasn’t able to bind a. proteins that did not get hit by tag were already targeted by OP 2. control proteome does not bind to enzyme; each enzyme is tagged by a probe; gel is continuous as each enzyme was tagged. Fungicides 1. Chlorothalonil (CTN) 1. Mode of Action a. Glutathione depletion b. “numerous enzyme targets” and a “multi-mode site of action” 2. Toxicity a. Probable human carcinogen, known animal carcinogen 2. Dithiocarbamate 1. Mode of action a. non-selective inhibition of sulfhydryl groups 2. Toxicity a. Linked to Parkinson’s disease b. endocrine disruption Herbicides 1. Atrazine 1. Mode of Action a. inhibits photosynthesis 2. Toxicity a. induces aromatase: promotes the conversion of testosteroneàestrogen b. may also lead to increases in reproductive cancers in humans 2. 2,4-Dichlorophenoxyaliphatic acid 1. Mode of Action a. plant growth hormone; “grow plant to death” 2. Toxicity a. Low toxicity to fish and wildlife b. Mammals – relatively safe, not carcinogenic 3. Similar structure to 2,4,5-T (Agent Orange) – unstable due to Dioxin 3. Glyphosate (RoundUp) 1. Mode of Action a. binds to and inhibits EPSPS, an enzyme essential for protein synthesis in plants 2. Toxicity a. Broad-spectrum nonselective for grasses, weeds and woody plants. GMOs vs Organic Foods 1. Why we Pest Control 1. Population and food a. Population increase with little change in available arable land and water 2. Pest losses must be reduced for maximum production a. 1/3 of world’s food crops are lost from pests during growth, harvesting, or storage 2. GMO Crops 1. RoundUp Ready: Overproduce EPSPS and GSH to overcome Glyphosate Toxicity 2. Bt Corn: contains gene from Bacillus thuringensis that produces Cry toxins that lyse insect guts Lecture 7: Gene Regulation of Xenobiotic Metabolic Enzymes 1. Regulation of Xenobiotic Metabolism 1. Induction of xenobiotic metabolism enzymes occurs at a genetic level 2. Regulation of gene expression 1. enhancer (cis-acting response element): a. DNA sequences that bind to specific transcription factors and enhance transcription b. e.g. estrogen response element, dioxin response element 2. transcription regulator (trans-acting factor): a. transcriptional cofactors that function in chromatin remodeling and recruitment of basal transcription complex 3. Promoter Bashing 1. reporter gene assay in combination with deletion and mutation introduce reporter genes into cells (transfection) à treat cells with compounds of interest (TCDD) à measure reporter gene activity in cells via luciferase after each deletion à compare which deletion reduces reporter gene activity With each deletion, you change the percent fold of luciferase activity. Your response element is most likely between 200 and 100 base pairs. 4. Mechanism of Transfection: introducing DNA into cells 1. viral transfection: via plasmid 2. mikro injection: directly introduce DNA into nucleus 3. electroporation: electric charge to open up membrane and allow DNA to transfect 4. lipofection: uptake via endocytosis 5. Gel Shift/Bandshift Assay DNA alone is lighter and will travel further down the gel DNA+radiolabeled receptor probe is heavier and will migrate slower in gel 6. AhR Mediated Activation 1. AhR exists in the cytoplasm in association with chaperones (HSP90) a. Chaperone complex optimizes ligand binding (e.g. TCDD) 2. Upon ligand binding, AhR dissociates from HSP90 and translocates to the nucleus 3. AhR forms a heterodimer with Arnt 4. AhR/Arnt binds the XRE/DRE in the CYP promoter region to activate gene transcription 5. AhR naturally occurring ligands: bilirubin, tryptophan 6. AhR synthetic ligands: TCDD, PAHs 7. There is no endogenous ligand for AhR 7. AhR mediates Carcinogenesis 1. AhR induces tumor growth by inducing expression of CYPs 2. AhR +/+: a. BaP injections induce subcutaneous tumor growth 3. AhR -/-: a. resistant to BaP-induced tumors 8. AhR mediates TCDD toxicity 1. AhR mediates TCDD toxicity by inducing CYPs 2. AhR +/+: a. exposure to TCDD during development induces impaired renal development 3. AhR -/-: a. completely resistant to TCDD-induced effects Lecture 8: Nuclear Receptors and Toxicology 1. Nuclear Receptors 1. proteins found within cells that sense the presence of hormones and certain lipids 2. human genome has 48 NHRs 3. 3 types: a. endocrine receptors: hormonal lipids b. adopted orphan receptors: dietary lipids c. orphan receptors: unknown 4. Structural Organization DBD: contains two zinc fingers that make contacts with DNA LBD: binds to specific lipophilic molecules A/B and F: transactivation domains; F activity is dependent on ligands 2. Type 1 Nuclear Receptors 1. Steroid Receptors: glucocorticoids, androgen, progesterone, estrogen 2. Mode of Action a. receptor is located in cytoplasm bound to hsp90 i. chaperone complexes are required for optimal steroid hormone binding b. ligand binding allows receptor to dissociate from hsp90 complex and enter the nucleus c. nuclear receptor homodimer binds to the Hormone Response Element (HRE) d. regulates gene expression 3. Experiment: Fractions with GR and no hsp90 have very low radioactivity Fractions with hsp90 have much greater radioactivity Lane 2: GR + unfractionated lysate Lane 9: GR +A,B,C 4. Type 2 Nuclear Receptors 1. Examples: CAR, PPAR, thyroid, retinoids 2. Mode of Action a. receptor is located on the nucleus bound to transcriptional corepressors i. transcriptional corepressors suppress gene transcription b. ligand binding releases the corepressor and recruits coactivators i. the ligand and the response element affect the conformation of receptors and the interaction with coregulators c. nuclear receptor/coactivator heterodimer binds to HRE d. regulate gene expression 5. Agonist 1. ligand that binds to a receptor à recruits coactivatorsà gene transcription 6. Antagonist 1. ligand binds to the receptor à prevents c
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