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

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


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


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

pyruva

te deh

ydrogen

ase 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


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


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)

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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


7

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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8

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

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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)

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Hemoglobin: Aplastic Anemia aplastic marrow normal marrow

www.hopkinsmedicine.org 27


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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


11

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

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12

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

!  NADH-dependent reductase (Cytochrome b5 reduc) !  NADPH-dependent reductase (intact HMP, G6PD) !  Catalase !  Hydrogen peroxidase

35

Hemoglobin: Methemoglobin

!  Fe2+ state: deoxyhemoglobin carries oxygen !  Fe3+ state: methemoglobin results from

oxidation and does not carry hemoglobin

HgbFe2+ + O2 → HgbFe3+O2 → HgbFe2+ + O2 > HgbFe3+ + O2-

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!  Cyt b5 or NADH dependent reductase !  NADPH-dependent reductase

HgbFe2+

HgbFe3+

cyt b5

cyt b5+ NADH

NAD+

Cyt b5 reductase

MB+

LMB

NADPH

NADP+

NADPH- dep reductase

glycolysis hexose monophosphate shunt 37


!  Chemicals !  aniline !  arsine !  chlorates (old strike matches) !  chlorobenzene !  copper sulfate !  napthalene !  nitrites (food contaminants & poppers) !  nitrates (food & well water) !  NOx (oxides of nitrogen) !  phenol

aniline

chloro- & nitrobenzene

phenol 38


!  Medications !  -caines (benzo-, lido-, prilo-) !  dapsone (sulfonamide deriv) !  methylene blue !  nitrites, nitrates !  phenacetin !  phenazopyridine !  -quines (chloro-, prima-) !  sulfonamide antibiotics

aniline

sulfanilamide

phenacetin

benzocaine

39


14

Hemoglobin: Sulfhemoglobin

!  Characteristics !  Same agents that induce methgb !  Not reversible !  Shifts O2-hgb dissociation curve to right !  H2S arguable, probably does not cause

sulfhgb, and hopefully would not be a great test item.

40

vettech.vet.ku.edu

Heinz Body

41

Hemoglobin: Hemolysis !  Mechanism

!  Oxidation of the globin chain !  Glutathione depletion or membrane injury

!  Characteristics !  Extravascular: mild forms !  Intravascular: severe forms

!  Anemia !  Free hgb (in serum and urine) !  Reticulocytosis !  Decreased haptoglobin

ww

w.re

side

nts.

path

olog

y.pi

tt.ed

u

42


15

Hemoglobin: Hemolysis

!  Hemolysis > methemoglobin !  Arsine (AsH3) !  Stibine (SbH3) !  Napthalene !  Copper sulfate

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Hemoglobin: Non-oxidant, Immune-mediated Hemolysis

!  Type I (IgG-mediated) !  penicillin

!  Type IV (Cell-mediated) !  α-methyldopa

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Hemoglobin: Toxins

! Competition for oxygen binding ! COHgb ! MetHgb ! SHgb

45


16

1.2.5 Channels

!  Sodium Channels !  Calcium channels !  Potassium channels

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1.2.5 Sodium Channel

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Na+ Channel Structure

!  Found in neurons, glial cells & myocytes !  9 subtypes !  Tetrameric protein !  Transmembrane !  SCN gene

!  SCN5 – Brugada Syndrome !  Voltage-gated (myocardial) !  Ligand-gated (nicotinic)

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17

Na+ Channel Function

!  Resting (Closed) !  Open !  Inactivated (Closed)

!  Refractory to opening

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Myocardial Na+ Channel Function

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Na+ Channel Modulation

!  Agonists – channel openers !  Aconitine (Monk�s Hood) !  Batrachotoxin (Poison Dart Frog) !  Ciguatoxin !  Grayanotoxin (Azalea & Rhododendron) !  Veratridine (Hellebore sp)

51


18


!  Antagonists !  Ia Antidysrhythmics

!  Procainamide, quinidine, disopyramide !  Ib Antidysrhythmics

!  Lidocaine, phenytoin !  Ic Antidysrhythmics

!  Encainide, flecainide, propafenone !  Others

!  Amiodarone, carbamazepine, cocaine, diphenhydramine, propranolol, propoxyphene, thioridazine and metabolite

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Na in K out

Ca in, K out

53


54


19

Na+ Channel Modulation: Rate-Dependent baseline

7:50 min

2:50 min

QRS – 140 ms; BP - 145/78 mmHg

5:50 min

QRS – 160 ms; BP - 151/68 mmHg

QRS – 220 ms; BP - 0 mmHg QRS – 180 ms; BP -164/65 mmHg 55

Na Channel Recovery

!  Class Ia: τrecovery 1-10 sec !  Class Ib: τrecovery < 1 sec !  Class Ic: τrecovery > 10 sec

56

1.2.5 Ca2+ Channels

!  L-type !  N-type (neuronal) !  P-type (Purkinje) !  T-type (muscular)

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20

L-type Ca2+ Channel

!  Four proteins !  Span cell membranes !  Regulates calcium entry

!  Closed in resting state !  Require activation to open

!  Channel location determines the functional result of calcium entry

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L-type Ca2+ Channel

endocrine non-vascular smooth muscle 59

Ca2+ Channel Activation - Myocardial

!  Ca2+- mediated Ca2+ - release !  Result

!  ↑HR !  ↑contractility

!  Modulators !  Catecholamines !  G protein !  cAMP !  protein kinase

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21

Ca2+ Channel Activation - Vascular

!  Result !  vasoconstriction !  Maintenance of BP

!  Modulators !  α1 stimulation !  β2 stimulation !  angiotensin !  endothelin

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Ca2+ Channel Antagonism

Consequences: Hypotension Bradycardia Poor cardiac output Cardiogenic Shock

X

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Ca2+ Channel Antagonism

!  CCB drugs !  Nifedipine (dihydropyridine) !  Diltiazem (benzothiazepine) !  Verapamil (phenylalkylamine) !  Bepridil (diarylaminopropylamine)

!  Cyclic antidepressants !  Propafenone

63


22

Ca2+ Channel Agonist

!  Levosimendan !  Directly opens Ca2+ channel !  Heart failure treatment !  Experimental treatment of CCB toxicity !  No human overdose

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1.2.5 Potassium Channels

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K+ Channel Structure !  Tetrameric protein in the cell membrane

!  Central pore through which K+ flows

!  Normally closed !  Opening leads to K+ efflux from the cell

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K+ Channel Function !  Inhibition of cell function

!  Acts to prevent overuse of the cell !  Opening stimuli

!  ↓intracellular energy molecules (ATP) !  ↑intracellular Na+

!  ↑intracellular Ca2+

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ATP-Dependent K+ Channel

ins

ins

ins

ins ins

ins

K+-ATP

Ca2+ Ca2+

Ca2+

ATP ADP + Pi 68

ATP-Dependent K+ Channel K+

ins

ins

Ca2+ Ca2+ Ca2+

ATP ADP + Pi

K+

K+

K+ K+

ins ins

X 69


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ATP-K+ Channel Modulation

ins

ins

ins

ins ins

ins

K+ channel

Ca2+ Ca2+

Ca2+

Sulfonylurea (glipizide, glyburide)

ins ins

ins

70

ATP-K+ Channel Modulation

K+

Ca2+

G

ins ins

ins

ins ins

ins

somatostatin receptor

(octreotide)

71

+ mV

- mV

Normal Function: Myocardial K+ Channel

Na+

Ca2+

72


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Normal Function: Myocardial K+ Channel

!  Effective Refractory Period !  Depolarization not possible

!  Relative Refractory Period !  Depolarization possible with sufficient

electrical stimulus

RRP ERP

73

K+ Channel Modulation

!  K+ channel inhibition (Class III drugs) !  Prolongs action potential (phase 3) !  Equalizes refractoriness of ischemic and non-

ischemic tissues

X Na+!

74

Prolonged QTc / TdP !  Antidysrhythmic

!  Class I (quinidine and quinine) !  Class III (amiodarone, bretylium, dofetilide, ibutilide)

!  Antidepressants !  Serotonin agonists

!  Antihistamine !  terfenadine, astemizole

!  Antipsychotic !  haloperidol - butyrophenone !  thioridazine - phenothiazine !  sertindole - atypical

75


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Prolonged QTc / TdP !  GI agents

!  cisapride !  Metabolic

!  hypokalemia (diuretics) !  hypomagnesemia (diuretics)

!  Metals !  arsenic

76

www.torsades.org

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1.3 Cytotoxic Mechanisms

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1.3 Antimitotics

!  Mitosis !  Forming of identical daughter cells by replicating and

dividing the chromosomes !  Replication occurs centrally in the parent cytoplasm !  Spindle apparatus attach to the chromosomes

(metaphase) and pull them towards the centromere (anaphase) prior to completion of cell division

www.cancerquest.emory.edu

79

Antimitotics

!  Spindle apparatus is composed of tubulin !  polymerized subunits !  polymerization ↔ depolymerization

!  Antimitotics interfere with spindle function !  Inhibit polymerization

!  colchicine, vincristine, vinblastine (vinca alkaloids) !  Inhibit depolymerization

!  taxol (alkaloid from Yew) !  Result: metaphase arrest

80

1.3 Apoptosis: Programmed Cell Death

!  Homeostatic mechanism for removal of: !  damaged, infected, aged cells !  activated immune cells (no longer needed)

!  Non-inflammatory !  Extrinsic triggers !  Intrinsic triggers

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Apoptosis

Extrinisic !  TNF-R !  Fas (CD95) !  Death receptor 3-5

Intrinisic !  Nuclear p53 !  Mitochondrial cyt C

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Apoptosis

Results !  Caspase family activation (3,7,8,9,10)

!  Cleaves DNA, protein !  Apoptotic protein activation (BH3 family)

!  Initiates mitochondrial pore formation 83

Apoptosis: Programmed Cell Death

!  Progressive condensation of nuclear contents !  Nucleus lyses (karyorhexis) !  Cell shrinkage, cytoplasmic condensation !  Apoptotic bodies, �budding� formed !  Macrophages remove apoptotic bodies

Normal lymphocyte

apoptotic lymphocyte

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Apoptosis

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