Post on 26-Dec-2020
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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
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de
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tho
log
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itt.
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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