ACMT Board Review 2012: Molecular Mechanisms1.3 Apoptosis: Programmed Cell Death Homeostatic...

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ACMT Board Review 2012: Molecular Mechanisms Russ Kerns, MD, FACMT Carolinas Medical Center Charlotte, NC

Transcript of ACMT Board Review 2012: Molecular Mechanisms1.3 Apoptosis: Programmed Cell Death Homeostatic...

ACMT Board Review 2012: Molecular Mechanisms

Russ Kerns, MD, FACMT Carolinas Medical Center

Charlotte, NC

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

1.2.1 Glycolysis & Oxidative Phosphorylation

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.

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+

  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

  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

Glycolysis

CH3CO-CoA acetyl-CoA

NADH

CH3COCOOH

CoA, NAD+

CH3CH2OCOOH

NAD+

lactate

pyruvate

NADH, CO2

Glycolysis: Toxins

CH3CO-CoA acetyl-CoA

CH3COCOOH

lipoamide

pyruvate

dihydrolipoamide

As3+

X

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

Kreb’s TCA Cycle: Toxins

Rodenticides   Sodium monofluoroacetate   Fluoroacetamide

acetyl-CoA

fluorocitrate

isocitrate

oxaloacetate

cis-aconitate monofluoroacetate

fluoroacetamide

Electron Transport Chain   Mitochondrial process   Series of oxidation-reduction reactions

  Cytochrome enzymes   FADH2 and NADH electron sources   Produce H2O and ATP

ETC: Toxins   Enzyme inhibitors   Uncouplers

X X

cytochrome oxidase aa3 NADH-CoQ reductase

X

Cytochrome Oxidase Inhibitors

acetyl CoA

lactate

glucose

pyruvate

TCA cycle

ATP H+

e- transport X X X

lactate

lactate

lactate lactate

lactate lactate

lactate

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)

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

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

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

  ethanol   Hypoglycin(?)

  Carnitine   Valproate

  Undefined mechanism   Aflatoxin   Amiodarone   cereulide   dimethylformamide   tetracycline

www.australianprescriber.com

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

1.2.4 Transport Proteins

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

  protoporhyrin ring   central iron atom

Hemoglobin: Toxins

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

Heme Synthesis: Direct Toxin

Harrison’s On-Line

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

Heme Synthesis: AIP

  Barbiturates   Carisoprodol   Danazol   Ethchlorvinyl   Meprobamate   Primidone   Pyrazolones   Trimethadione

dark red urine

www.porphyriafoundation.com

Hemoglobin: Toxins

  Erythropoiesis  Nephrotoxins - ↓ erythropoietin  Pure rbc aplasia – rare

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

Hemoglobin: Aplastic Anemia aplastic marrow normal marrow

www.hopkinsmedicine.org

Hemoglobin: Aplastic Anemia

  Immune mediated   T lymphocytes release cytokines

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

TNF

interferon-γ

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

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

Hemoglobin: Aplastic Anemia   Chemicals

  Benzene, lindane   Metals

  Arsenic   Miscellaneous

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

  Radiation

Hemoglobin: Toxins

www.pathology.vcu.edu

Hemoglobin: Toxins

  Megaloblastic anemia   ↓ Vit B12 absorption

  Colchicine, metformin, neomycin   ↓ folate absorption

  Etoh   Impaired dihydrofolate reductase

  Methotrexate   Pyrimethamine   Pyridium   Trimethoprim

Hemoglobin: Oxidant Stress

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

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

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-

Hemoglobin: Methemoglobin

  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

Hemoglobin: Methemoglobin

  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

Hemoglobin: Methemoglobin

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

aniline

sulfanilamide

phenacetin

benzocaine

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.

vettech.vet.ku.edu

Heinz Body

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

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side

nts.

path

olog

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tt.ed

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Hemoglobin: Hemolysis

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

Hemoglobin: Non-oxidant, Immune-mediated Hemolysis

  Type I (IgG-mediated)   penicillin

  Type IV (Cell-mediated)   α-methyldopa

Hemoglobin: Toxins

 Competition for oxygen binding  COHgb  MetHgb  SHgb

1.2.5 Channels

  Sodium Channels   Calcium channels   Potassium channels

1.2.5 Sodium Channel

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)

Na+ Channel Function

  Resting (Closed)   Open   Inactivated (Closed)

  Refractory to opening

Myocardial Na+ Channel Function

Na+ Channel Modulation

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

Na+ Channel Modulation

  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

Na in K out

Ca in, K out

Na+ Channel Modulation

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

Na Channel Recovery

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

1.2.5 Ca2+ Channels

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

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

L-type Ca2+ Channel

endocrine non-vascular smooth muscle

Ca2+ Channel Activation - Myocardial

  Ca2+- mediated Ca2+ - release   Result

  ↑HR   ↑contractility

  Modulators   Catecholamines   G protein   cAMP   protein kinase

Ca2+ Channel Activation - Vascular

  Result   vasoconstriction   Maintenance of BP

  Modulators   α1 stimulation   β2 stimulation   angiotensin   endothelin

Ca2+ Channel Antagonism

Consequences: Hypotension Bradycardia Poor cardiac output

Cardiogenic Shock

Ca2+ Channel Antagonism

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

  Cyclic antidepressants   Propafenone

Ca2+ Channel Agonist

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

1.2.5 Potassium Channels

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

K+ Channel Function   Inhibition of cell function

  Acts to prevent overuse of the cell   Opening stimuli

  ↓intracellular energy molecules (ATP)   ↑intracellular Na+

  ↑intracellular Ca2+

ATP-Dependent K+ Channel

ins

ins

ins

ins ins

ins

K+-ATP

Ca2+ Ca2+

Ca2+

ATP ADP + Pi

ATP-Dependent K+ Channel K+

ins

ins

Ca2+ Ca2+ Ca2+

ATP ADP + Pi

K+

K+

K+ K+

ins ins

X

ATP-K+ Channel Modulation

ins

ins

ins

ins ins

ins

K+ channel

Ca2+ Ca2+

Ca2+

Sulfonylurea (glipizide, glyburide)

ins ins

ins

ATP-K+ Channel Modulation

K+

Ca2+

G

ins ins

ins

ins ins

ins

somatostatin receptor

(octreotide)

+ mV

- mV

Normal Function: Myocardial K+ Channel

Na+

Ca2+

Normal Function: Myocardial K+ Channel

  Effective Refractory Period   Depolarization not possible

  Relative Refractory Period   Depolarization possible with sufficient

electrical stimulus

RRP ERP

K+ Channel Modulation

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

ischemic tissues

X Na+!

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

Prolonged QTc / TdP   GI agents

  cisapride   Metabolic

  hypokalemia (diuretics)   hypomagnesemia (diuretics)

  Metals   arsenic

www.torsades.org

1.3 Cytotoxic Mechanisms

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

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

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

Apoptosis

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

Intrinisic   Nuclear p53   Mitochondrial cyt C

Apoptosis

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

  Cleaves DNA, protein   Apoptotic protein activation (BH3 family)

  Initiates mitochondrial pore formation

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

Apoptosis