Molecular Mechanisms

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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

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1.2.1 Glycolysis & Oxidative Phosphorylation

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Molecular Mechanisms

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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.

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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

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Molecular Mechanisms

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!  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

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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

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Glycolysis: Toxins

CH3CO-CoA acetyl-CoA

CH3COCOOH

lipoamide

pyruvate

pyruvate decarboxylase

dihydrolipoamide

As3+

thiamine

X

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Molecular Mechanisms

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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

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Kreb s TCA Cycle: Toxins

Rodenticides !  Sodium monofluoroacetate !  Fluoroacetamide

acetyl-CoA

fluorocitrate

isocitrate

oxaloacetate

cis-aconitate monofluoroacetate

X

fluoroacetamide

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

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

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Molecular Mechanisms

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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

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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)

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Molecular Mechanisms

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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

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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

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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

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

!  protoporhyrin ring !  central iron atom

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Molecular Mechanisms

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Hemoglobin: Toxins

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

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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

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Molecular Mechanisms

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Heme Synthesis: AIP

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

dark red urine

www.porphyriafoundation.com

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Hemoglobin: Toxins

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

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

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Hemoglobin: Aplastic Anemia aplastic marrow normal marrow

www.hopkinsmedicine.org 27

Molecular Mechanisms

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Hemoglobin: Aplastic Anemia

!  Immune mediated !  T lymphocytes release cytokines

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

TNF

interferon-γ

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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

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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

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Hemoglobin: Toxins

!  Megaloblastic anemia !  ↓ Vit B12 absorption

!  Colchicine, metformin, neomycin !  ↓ folate absorption

!  Etoh !  Impaired dihydrofolate reductase

!  Methotrexate !  Pyrimethamine !  Pyridium !  Trimethoprim

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Molecular Mechanisms

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Hemoglobin: Oxidant Stress

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

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Hemoglobin: Oxidant Stress

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

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