ACMT Board Review 2012: Molecular Mechanisms ... Molecular Mechanisms 1 ACMT Board Review 2012:...

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  • Molecular Mechanisms

    1

    ACMT Board Review 2012: Molecular Mechanisms

    Russ Kerns, MD, FACMT Carolinas Medical Center

    Charlotte, NC 1

    Objectives: Cover Core Content

    !  1.2 Molecular components/mechanisms !  1.2.1 Glycolysis & oxidative phosphorylation !  1.2.2 Other metabolic pathways (β-oxidation) !  1.2.4 Transport proteins (hemoglobin) !  1.2.5 Channels

    !  1.3 Cytotoxic mechanisms !  Provide key example toxins

    2

    1.2.1 Glycolysis & Oxidative Phosphorylation

    3

  • Molecular Mechanisms

    2

    Oxidative Phosphorylation

    !  Energy is released when ATP → P + ADP !  Restoration of ATP (energy stores) involves

    phosphorylation of ADP via coupling of oxidation of H+ to form H20.

    4

    Oxidative Phosphorylation

    carbohydrate/fatty acid

    acetyl-CoA

    TCA

    NADH/FADH2

    electron transport chain

    H+ + e-

    P

    ADP ATP H2O ADP antiporter

    H+

    e- + O2- + H+

    ATP synthase

    H+ H+ 5

    !  Main carb metab path !  Cytosolic process !  6C cmpd → 2 X 3 C cmpd !  Net 2 ATP molecules !  Make pyruvate for Kreb s

    Glycolysis glucose

    glucose-6-P

    fructose-6-P

    glyceraldehyde-3-P + DHA-P

    2 [pyruvate]

    fructose-1,6-diP

    2 [glyceraldehyde-3-P]

    2 [1,3-diphosphoglycerate]

    2 [P intermediates]

    ATP

    ATP

    ATP

    ATP

    6

  • Molecular Mechanisms

    3

    !  Arsenic (V) !  Substitutes for P !  Fail to make 1,3-diP intermed !  Fail to make ATP !  Fail to make pyruvate

    Glycolysis: Toxins

    glyceraldehyde-3-P + DHA-P

    2 [glyceraldehyde-3-P]

    2 [1,3-diphosphoglycerate]

    2 [P intermediates]

    ATP

    2NAD+ + 2 P As5+ X

    7

    Glycolysis

    CH3CO-CoA acetyl-CoA

    NADH

    CH3COCOOH

    py ruv

    ate de

    hy dro

    ge na

    se CoA, NAD+

    CH3CH2OCOOH

    NAD+

    lactate

    pyruvate

    pyruvate decarboxylase

    NADH, CO2 thiamine

    8

    Glycolysis: Toxins

    CH3CO-CoA acetyl-CoA

    CH3COCOOH

    lipoamide

    pyruvate

    pyruvate decarboxylase

    dihydrolipoamide

    As3+

    thiamine

    X

    9

  • Molecular Mechanisms

    4

    Kreb s TCA Cycle pyruvate acetyl-CoA

    NADH NAD+

    citrate

    Succinyl-CoA

    oxalosuccinate

    isocitrate

    oxaloacetate

    succinate

    maleate cis-aconitate

    α-ketoglutarate

    fumarate

    NAD+

    NADH

    NAD+ NADH

    FADH FADH2

    10

    Kreb s TCA Cycle: Toxins

    Rodenticides !  Sodium monofluoroacetate !  Fluoroacetamide

    acetyl-CoA

    fluorocitrate

    isocitrate

    oxaloacetate

    cis-aconitate monofluoroacetate

    X

    fluoroacetamide

    11

    Electron Transport Chain !  Mitochondrial process !  Series of oxidation-reduction reactions

    !  Cytochrome enzymes !  FADH2 and NADH electron sources !  Produce H2O and ATP

    12

  • Molecular Mechanisms

    5

    ETC: Toxins !  Enzyme inhibitors !  Uncouplers

    X X

    cytochrome oxidase aa3 NADH-CoQ reductase

    X 13

    Cytochrome Oxidase Inhibitors

    acetyl CoA

    lactate

    glucose

    pyruvate

    TCA cycle

    ATP H +

    e- transport X X X

    lactate

    lactate lactate

    lactate

    lactate lactate

    lactate

    14

    ETC: Toxins !  Uncouplers

    !  Salicylate !  Dinitrophenol (explosives & wood preservative) !  Pentachlorophenol (fungicide)

    !  Cytochrome aa3 inhibitors !  Cyanide !  H2S !  CO !  Methanol !  Phosphine gas !  Sodium azide (propellant in airbags)

    !  NADH-CoQ reductase !  Rotenone (plant derived fish poison)

    15

  • Molecular Mechanisms

    6

    1.2.2 β-Oxidation of FFA

    fatty acyl-CoA

    CoA carnitine

    fatty acylcarnitine fatty acyl-CoA

    carnitine-palmitoyltransferase

    CoA

    (CH)nCO-CoA (CH)n-2CO-CoA + CH3CO-CoA acetyl-CoA

    NADH, FADH2

    CoA

    (CH)nCOOH (CH)nCO-CoA acyl-CoA synthetase

    ATP

    fatty acyl-CoA

    ADP + P CoA

    16

    β-Oxidation of FFA: Toxins

    fatty acyl-CoA

    CoA carnitine

    fatty acylcarnitine fatty acyl-CoA

    CoA

    (CH)nCO-CoA (CH)n-2CO-CoA + CH3CO-CoA acetyl-CoA

    NADH, FADH2

    CoA

    etoh, hypoglycin

    valproate

    17

    β-Oxidation of FFA: Toxins !  ↑ NADH/NAD+ ratio

    !  ethanol !  Hypoglycin(?)

    !  Carnitine !  Valproate

    !  Undefined mechanism !  Aflatoxin !  Amiodarone !  cereulide !  dimethylformamide !  tetracycline

    www.australianprescriber.com 18

  • Molecular Mechanisms

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    Mitochondrial DNA: NRTIs

    !  Nucleoside reverse transcriptase inhibitors !  Mechanism

    !  Inhibit mitochondrial DNA replication !  Inhibit ADP/ATP antiporter(?)

    !  Result !  Lactic acidosis ± steatosis

    !  Agents !  Stavudine !  Didandosine !  Zalcitobine !  Zidovudine !  Tenofovir (nucleotide)

    X 19

    1.2.4 Transport Proteins

    20

    Hemoglobin !  iron-based tetrameric protein !  α- and β-globin chains (2 each) !  Heme complex in each chain (4 total)

    !  protoporhyrin ring !  central iron atom

    21

  • Molecular Mechanisms

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    Hemoglobin: Toxins

    ! Site of action of toxins ! Heme synthesis ! Erythropoiesis ! Hemorrhage ! Oxidant stress ! Competition for oxygen binding

    22

    Heme Synthesis: Direct Toxin

    Harrison s On-Line 23

    Heme Synthesis: Indirect Toxins

    !  Acute Intermittent Porphyria !  Hepatic !  Autosomal dominant !  Reduced HMB synthase activity !  Some drugs may exacerbate AIP by

    increasing ALA-synthase activity !  Poorly defined mechanism

    24

  • Molecular Mechanisms

    9

    Heme Synthesis: AIP

    !  Barbiturates !  Carisoprodol !  Danazol !  Ethchlorvinyl !  Meprobamate !  Primidone !  Pyrazolones !  Trimethadione

    dark red urine

    www.porphyriafoundation.com

    25

    Hemoglobin: Toxins

    !  Erythropoiesis ! Nephrotoxins - ↓ erythropoietin ! Pure rbc aplasia – rare

    ! INH ! Hypoglycemics (chlorpropamide, tolbutamide) ! Phenytoin ! Sulfasalazine ! Valproate (single case report)

    26

    Hemoglobin: Aplastic Anemia aplastic marrow normal marrow

    www.hopkinsmedicine.org 27

  • Molecular Mechanisms

    10

    Hemoglobin: Aplastic Anemia

    !  Immune mediated !  T lymphocytes release cytokines

    !  Suppress hematopoietic stem cells !  Apoptosis (↑ Fas receptors on stem cells)

    TNF

    interferon-γ

    28

    Hemoglobin: Aplastic Anemia

    !  DNA injury !  Direct DNA injury

    !  Ionizing radiation !  Inhibition of DNA replication

    !  Folate inhibitors (methotrexate) !  Intermediary metabolite that binds DNA

    !  Benzene (quinone + free radicals) !  Tubulin inhibition during cell replication

    !  Antimitotics (colchicine, vincristine, vinblastin)

    metaphase arrest

    metaphase.wordpress.com

    29

    Hemoglobin: Aplastic Anemia !  Antibiotics

    !  Chloramphenicol !  Anti-convulsants

    !  Carbamazepine, phenytoin !  Anti-inflammatory agents

    !  Diclofenac, D-penicilamine, gold salts, indomethicin, phenylbutazone

    !  Anti-neoplastic agents !  Alkylating agents (nitrogen mustards) !  Antibiotics (danorubicin, adriamycin) !  Antimitotics (colchicine, vinblastin, vincristine) !  Antimetabolites (purine and pyrimidine analogues)

    !  Antipsychotics !  Chlorpromazine, clozapine 30

  • Molecular Mechanisms

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    Hemoglobin: Aplastic Anemia !  Chemicals

    !  Benzene, lindane !  Metals

    !  Arsenic !  Miscellaneous

    !  Acetazolamide, captopril, cimetidine, chlorpromazine, dapsone, fluoxetine, meprobamate, nifedipine, PTU, ticlopidine, tocainide

    !  Radiation 31

    Hemoglobin: Toxins

    www.pathology.vcu.edu

    32

    Hemoglobin: Toxins

    !  Megaloblastic anemia !  ↓ Vit B12 absorption

    !  Colchicine, metformin, neomycin !  ↓ folate absorption

    !  Etoh !  Impaired dihydrofolate reductase

    !  Methotrexate !  Pyrimethamine !  Pyridium !  Trimethoprim

    33

  • Molecular Mechanisms

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    Hemoglobin: Oxidant Stress

    !  Heme: deoxyhgb → Methgb (Fe2+→Fe3+) !  Prophyrin ring by sulfur: Sulfhgb !  Globin: Heinz body hemolytic anemia

    34

    Hemoglobin: Oxidant Stress

    !  Protection from oxidant stress !  Ascorbic acid (Vit C) !  Glutathione (intact HMP, G6PD) !  Enzymatic

    !