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ACMT Board Review 2014:Molecular Mechanisms

Handout

Russ Kerns, MD, FACMT

Carolinas Medical Center & Carolinas Poison 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

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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.

Oxidative Phosphorylation

carbohydrate/fatty acid

acetyl-CoA

TCA

NADH/FADH2

electron transport chain

H+ + e-

P

ADP

ATPH2O

ADPantiporter

H+

e- + O2- + H+

ATPsynthase

H+ H+

Main carb metab path

Cytosolic process

6C cmpd → 2 X 3 C cmpd

Net 2 ATP molecules

Make pyruvate for Kreb’s

Glycolysisglucose

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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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-CoAacetyl-CoA

NADH

CH3COCOOH

CoA, NAD+

CH3CH2OCOOH

NAD+

lactate

pyruvate

NADH, CO2

Glycolysis: Toxins

CH3CO-CoAacetyl-CoA

CH3COCOOH

lipoamide

pyruvate

dihydrolipoamide

As3+

X

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Kreb’s TCA Cyclepyruvate acetyl-CoA

NADHNAD+

citrate

Succinyl-CoA

oxalosuccinate

isocitrate

oxaloacetate

succinate

maleate cis-aconitate

α-ketoglutarate

fumarate

NAD+

NADH

NAD+NADH

FADHFADH2

Kreb’s TCA Cycle: Toxins

Rodenticides

Sodium monofluoroacetate

Fluoroacetamide

acetyl-CoA

fluorocitrate

isocitrate

oxaloacetate

cis-aconitatemonofluoroacetate

fluoroacetamide

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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ETC: Toxins Enzyme inhibitors Uncouplers

XX

cytochrome oxidase aa3

NADH-CoQ reductase

X

Cytochrome Oxidase Inhibitors

acetyl CoA

lactate

glucose

pyruvate

TCA cycle

ATPH+

e- transportXX X

lactate

lactate

lactatelactate

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)

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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-CoAacetyl-CoA

NADH, FADH2

CoA

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

ATP

fatty acyl-CoA

ADP + PCoA

β-Oxidation of FFA: Toxins

fatty acyl-CoA

CoA carnitine

fatty acylcarnitine fatty acyl-CoA

CoA

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

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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

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

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Hemoglobin: Toxins

Site of action of toxinsHeme 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

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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

INHHypoglycemics (chlorpropamide, tolbutamide)PhenytoinSulfasalazineValproate (single case report)

Hemoglobin: Aplastic Anemiaaplastic marrow normal marrow

www.hopkinsmedicine.org

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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

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

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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

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

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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-

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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

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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

w.r

esi

de

nts

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tho

log

y.p

itt.

ed

u

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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 bindingCOHgbMetHgbSHgb

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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)

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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)

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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

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Na+ Channel Modulation:Rate-Dependentbaseline

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 mmHgQRS – 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)

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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

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Ca2+ Channel Activation - Vascular

Result vasoconstriction Maintenance of BP

Modulators α1 stimulation β2 stimulation angiotensin endothelin

Ca2+ Channel Antagonism

Consequences:HypotensionBradycardiaPoor cardiac output

Cardiogenic Shock

X

Ca2+ Channel Antagonism

CCB drugs Nifedipine (dihydropyridine)

Diltiazem (benzothiazepine)

Verapamil (phenylalkylamine)

Bepridil (diarylaminopropylamine)

Cyclic antidepressants

Propafenone

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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

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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+

ATP-Dependent K+ Channel

ins

ins

ins

insins

ins

K+-ATP

Ca2+Ca2+

Ca2+

ATP ADP + Pi

ATP-Dependent K+ ChannelK+

ins

ins

Ca2+

Ca2+

Ca2+

ATP ADP + Pi

K+

K+

K+K+

insins

X

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ATP-K+ Channel Modulation

ins

ins

ins

insins

ins

K+ channel

Ca2+

Ca2+

Ca2+

Sulfonylurea (glipizide, glyburide)

insins

ins

ATP-K+ Channel Modulation

K+

Ca2+

GG

insins

ins

insins

ins

somatostatinreceptor

(octreotide)

+ mV

- mV

Normal Function:Myocardial K+ Channel

Na+

Ca2+

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Normal Function:Myocardial K+ Channel

Effective Refractory Period Depolarization not possible

Relative Refractory Period Depolarization possible with sufficient

electrical stimulus

RRPERP

K+ Channel Modulation

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

ischemic tissues

XNa+

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

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Prolonged QTc / TdP

GI agents cisapride

Metabolic hypokalemia (diuretics)

hypomagnesemia (diuretics)

Metals arsenic

www.torsades.org

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

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

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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

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Apoptosis