CARDIAC TOXICOLOGY James Swenberg University of North Carolina.
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Transcript of CARDIAC TOXICOLOGY James Swenberg University of North Carolina.
CARDIAC TOXICOLOGY
James Swenberg
University of North Carolina
Basic Cardiovascular Function:
The heart, in concert with elastic blood vessels, maintains precise control of blood pressure and critical tissue perfusion
Cardiac Myocyte
Basic Myocardial Structure
Cardiac Tissue
Cardiac Myofibril
Cardiac Myofilaments
Basic Myocardial Structure• Cardiac muscle is striated, BUT :
– single nucleus – under autonomic control – all cardiac cells contract together
(skeletal - selective recruitment of motor units)
– high oxygen demand – structure permits coordinated contraction
(acts as a syncytium)– Cardiac myocytes do not regenerate
Superior Vena Cava Left Atrium
Bundle Branches
Papillary Muscle
Sinoatrial Node
Right Atrium
Atrioventricular Node
Right VentriclePurkinje Fibers
Purkinje Fibers
Left Ventricle
Bundle of His
Basic Cardiac Electrophysiology
Ion Channels and Transporters in Cardiac Myocytes
Na+ in fast
Decrease in voltage triggers Ca2+ in, K+ out, and Na+ out
Ca2+ in slow
K+ out fast
mitochondrion
Cellular Adaptation
Normal Cardiac Myocyte
Cell Death
Irreversible Cell Injury
Adapted Cell (hypertrophy)
Reversible Cell Injury
Figure adapted from Robbins Pathologic Basis of Disease, Cotran, et al.
Cell Hypoxia
Cardiac Myocyte Adaptation
Normal myocardium Myocardial degeneration
Myocardial necrosisCardiomyopathy
Hypertrophic Cardiomyopathy
Ischemic Injury
Manifestations of Cardiac Dysfunction
• Arrhythmias (flutters, fibrillations)
• Cardiomyopathies
• Organism effects– poor tissue oxygenation/perfusion (heart,
other)– accumulation of body fluids in inappropriate
locations (lungs, abdominal cavity, legs)– organ failure (kidney, liver, lungs)– in the extreme …….death
Measures of Toxicity:Alterations in Cardiovascular Function
• Physiologic function– Electrocardiograms (EKG)
• most sensitive early indicator of cardiac toxicity
– Heart rate• tail cuff method with photosensor (noninvasive)• implanted telemetry devices
– Systemic arterial blood pressure/blood flow• electromagnetic or doppler ultrasonic techniques
– Cardiac output• transthoracic echocardiography
Measures of Toxicity:Alterations in Cardiovascular Function
• Clinical Chemistry– Electrolyte disturbances/imbalances
• sodium, potassium, calcium, magnesium, zinc
– Blood gas (acid/base balance)– Proteins
• plasma albumin, myoglobin, fibrinogen
– Lipids• plasma total cholesterol and triglycerides• plasma lipoproteins, total lipids, phospholipids
Measures of Toxicity:Alterations in Cardiovascular
Structure• Clinical Chemistry
– enzyme release (short half-life)– creatine phosphokinase (CPK)
• hybrid dimer specific for cardiac muscle (CPK-MB)
– lactate dehydrogenase (LDH) -hydroxybutyrate dehydrogenase (-HBDH)– serum glutamic-oxaloacetic transaminase
(SGOT, AST)
Measures of Toxicity:Alterations in Cardiovascular
Function
• Anatomic Pathology– Direct cardiotoxicity– Thrombosis & infarction– Inflammation (myositis, endocarditis,
vasculitis)– Downstream tissue/organ effects
Compound-Induced Toxicity
• Toxicants that alter aerobic metabolism
• Toxicants that alter myocardial conduction
• Toxicants that alter cell membrane function
• Toxicants that directly damage myocardium
• Toxicants that induce vascular changes
Toxicants That Interfere With Aerobic Metabolism
• High energy demands of the heart make it susceptible to toxicants that interfere with:
– oxygen availability (e.g., nitrite, carbon monoxide)
– carbohydrate metabolism (e.g., fluoroacetate), or
– oxidative phosphorylation (e.g., dinitrophenols)
• rotenone
• antimycin A
• cyanide and carbon monoxide
• Toxicity may result in myocardial necrosis
Direct Cardiotoxicity: Myocardial Degeneration & Myocytolysis
Toxicant Interference With Oxidative Phosphorylation
RotenoneX
Antimycin AX
Cyanide and carbon monoxide
X
Toxicants That Alter Myocardial Conduction
• Alter impulse formation and cause arrhythmias– Toxicants that cause acidosis and hyperkalemia
(e.g., ethylene glycol)• enhance slow current activity• increase automaticity and promote arrhythmia
– Cardiotoxic divalent ions (e.g., barium, strontium)• replace calcium in slow-current channels• alter efflux of potassium from myocardial cells
hypokalemia and arrhythmias
Toxicants That Alter Myocardial Conduction
• Alter impulse formation and cause arrhythmias– Toxicants that cause prolongation of the QT
interval (e.g., seldane)
– Blockage of multiple ionic channels that may lead to syncope and ventricular fibrillation (torsade de pointes)
PQ S
R
TU
QT Interval
Toxicants That Cause Prolongation of the QT Interval
• Over 100 marketed pharmaceutical agents cause interference in ventricular repolarization
• QT prolongation is mentioned in the FDA-approved labeling as a known action of the drug
• e.g.Terfenadine (Seldane®) – antihistamine/removed in 1997Chlorpromazine (Thorazine®) – anti-psychoticArsenic trioxide (Trisenox®) – anti-cancer/leukemiaErythromycin (Erythrocin®) – antibioticFluoxetine (Prozac®, Sarafem®) – anti-depressant Haloperidol (Haldol®) – anti-psychotic/schizophrenia
Toxicants That Alter Myocardial Conduction
• Alter impulse formation and cause arrhythmias– Halogenated hydrocarbons (e.g., chloroform)
• suppress SA node (AV node becomes pacemaker)• sensitizes myocardium to arrhythmogenic effects of
sympathomimetic amines (catecholamines)
– Cardiac glycosides (e.g., digitalis)• inhibit the sodium-potassium exchange mechanism
decreased intracellular potassium, increased intracellular sodium catecholamine sensitivity
• increase refractory period of the AV node
Toxicants That Alter Cell Membrane Function
• Alter cell membrane control of ion movement and affect cardiac contraction– Cardiac glycosides and catecholamines– Chemical ionophores (e.g., monensin)
• facilitates the passage of sodium, potassium, or calcium• monensin: alters Ca2+ and Na+ transport increased
intracellular calcium changes myocardial contractility• excessive calcium accumulation impairs mitochondrial
oxidative phosphorylation myocardial necrosis
Toxicants That Alter Cell Membrane Function
• Alter cell membrane control of ion movement and affect cardiac contraction– Toxicants that bind to phospholipids (e.g.,
gossypol)• effect potassium transport hyperkalemia
arrhythmias
– Toxicants that selectively block sodium channels• tetrodotoxin, saxitoxin• decreased intracellular Na+ depression of normal
pacemaker function and conduction arrhythmias
Toxicants That Directly Damage Myocardium
• Damage the pumping effectiveness by reducing the number of active myocytes– Toxicants that cause oxidative damage and
lipid peroxidation (e.g., doxorubicin, ethanol)• redox cycling of doxorubicin semiquinone and
superoxide radicals
• ethanol metabolism lipid peroxidation of myocytes
• results in cell swelling, altered Ca2+ homeostasis, and irreversible myocyte injury
Toxicants That Directly Damage Myocardium
• Damage the pumping effectiveness by reducing the number of active myocytes– Toxicants that cause sarcolemmal injury and
calcium alterations (e.g., catecholamines)• endogenous: epinephrine and norepinephrine
• exogenous: isoproterenol (> toxicity than above)
• sarcolemmal damage through lipid peroxidation
• increased calcium uptake impaired mitochondrial function and activation of neutral proteases and phospholipases myocyte dysfunction and toxicity
Cardiovascular changes following chronic rodent exposure to
dioxin-like compounds
Reference: Jokinen MP, Walker NJ, Brix AE, Sells DM , Haseman JK, Nyska A (2003). Cardiovascular Pathology
in Female Sprague-Dawley Rats Following Chronic Treatment with Dioxin-like Compounds. Cardiovascular
Toxicology. ; 3(4): 299-310
Cardiomyopathy
• Myocardial fiber degeneration/necrosis, inflammation, fibrosis
• Common spontaneous degenerative change of myocardium in old rats– Cause unknown but is affected by diet and
stress
Incidences of Cardiomyopathy
• TCDD
• PCB126
Dose
(ng/kg)0 3 10 22 46 100 100
stop
10 12 22 25 32 36 22
Dose
(ng/kg)0 30 100 175 300 550 1000 1000
stop
9 16 17 16 24 28 32 15
Normal heart
Cardiomyopathy
Cardiac Toxicity observed in90-days exposure to Bis(2-
chloroethoxy)methane in rats and mice
Bis (2-chloroethoxy)methane (CEM) is a synthetic organic compound used as the
starting compound to produce polysulfide elastomers used extensively in a variety of
sealant applications.
Histopathologic definitions of cardiac lesions
Myocyte vacuolization
- Widespread accumulation of multiple, round, variably sized, primarily small, and clear vacuoles, located within the myocyte sarcoplasm
- Vacuoles, often similar in appearance, present in the interstitium are interpreted as a background change
Histopathologic definitions of cardiac lesions (Cont.)
Myocyte necrosis - Small areas containing fragmented,
angular, brightly eosinophilic myofibers with dark, shrunken nuclei
Atrial thrombosis - A mature, antemortem blood clot present
within the lumen of the atrium, consisting of alternating areas of fibrin and layered cellular elements
Dose (mg/kg/day)Histopathological findings
0 50 100 200 400 600 *
Males
Cardiomyopathy 10(1) 8(1.3) 10(1) 10(1) 1(1) 1(1)
Fibrosis 0 0 0 0 0 1(1)
Infiltration cell, mononuclear 0 0 1(1) 0 7(1) 10(1.8)
mineralization 1(1) 0 0 0 0 0
Myocardium, necrosis 0 0 0 0 0 7(3.7)
Myocardium, vacuolization, cytoplasmic
0 0 0 0 6(1.3) 9(1.9)
Atrium - thrombosis 0 0 0 0 0 3(1.4)
Females
Cardiomyopathy 8 (1) 6(1) 8 (1) 4(1) 3(1) 0
Infiltration cell, mononuclear 0 1(1) 0 7(1) 6(1.2) 10(2)
mineralization 0 0 0 1(1) 0 0
Myocardium, necrosis 0 0 0 1(1) 1(1) 6(1.7)
Mypcardium, vacuolization, cytoplasmic
0 0 0 2(1) 5(1.4) 8(1.6)
Histopathological findings in the heart in rats treatedfor 3 months with CEM (n=10). ( ) severity
* all male and female animals treated with the 600 mg/kg/day and two females treated with the 400 mg/kg/day died before the end of the study.
Atrial thrombosis
RR
MMLL
Heart of a female rat treated with 600 mg/kg of CEM (R-right ventricle; M-interventricular septa; L- left ventricle)
Myofiber vavuolation and mononuclear cell infitration
Myofiber cytoplasmic vacuolization
16-day cardiac toxicity of bis(2-chlorethoxy)methane H&E staining
ControlControl 2-D2-D
3-D3-D 5-D5-D
16-day cardiac toxicity of bis(2-chloroethoxy)methane masson’s trichrome staining
ControlControl 2-D2-D
3-D3-D 5-D5-D
16-day cardiac toxicity of bis(2-chlorethoxy)methane troponin immunostaining
ControlControl 2-D2-D
3-D3-D 5-D5-D
16-day cardiac toxicity of bis(2-chlorethoxy)methane TUNEL immunostaining
ControlControl 2-D2-D
3-D3-D 5-D5-D
Apoptotic signals
CI – CH2 – CH2 – O – CH2 – O – CH2 – CH2 – CIBis(2-chloroethoxy)methane
HOOC – CH2 – S – CH2 – COOHThiodiglycolic acid
Mitochondrial damage
Damage to myocytes - apoptosis
Cell death
Proposed Mechanism of Heart Toxicity
MITOCHONDRIAL CARDIOMYOPATHY IN 3'AZIDO-3'DEOXYTHYMIDINE (AZT)/ 3TC MULTIGENERATIONAL REPRODUCTIVE
ASSESSMENT BY CONTINUOUS BREEDING WHEN ADMINISTERED TO CD-1® MICE BY
GAVAGE AZT (Zidovudine) and 3TC
(Lamivudine) – are nucleoside reverse transcriptase inhibitors for
HIV-1 infection and AIDS-
AZT - Introduction• AZT is known to cause mitochondrial
myopathy in human and animals
• The mechanism of cardiomyopathy from AZT is not completely understood, but suggested to be related to depletion mtDNA replication, resulting in impaired synthesis of mitochondrial enzymes that generate ATP
- The enzyme responsible for mtDNA replication is DNA polymerase gamma, and it was found to be inhibited by AZT
Pathological changes in the heart
• Light microscopy – not commonly seen. Using masson’s trichrome – granular cytoplasm of myofibers, but no interstitial inflammation or fibrosis
• EM – Mitochondrial swelling, with fractured, dissolution and disrupted cristae (abnormal mitochondria is defined when there is loss or dissolution of more than 25% of the cristae area)
• Clinical chemistry – increased plasma lactate (lactic acidosis - indicating disturbed oxidative metabolism)
Changes in the heart of rats exposed to Ephedrine + Caffeine
Nyska A, Murphy E, Foley JF, Collins BJ, Petranka J, Howden R, Hanlon P, Dunnick JK. Acute Hemorrhagic Myocardial Necrosis and Sudden Death of Rats Exposed to a Combination of Ephedrine and Caffeine.Toxicol Sci. 2004 Nov 10
Ephedra ('ma huang‘)• An herbal dietary supplement for weight loss or
enhanced athletic performance• Currently a matter of national controversy. • At the heart of the debate are three important
questions: (1) The identity and composition of Ephedra
products with regard to ephedrine and related alkaloids;
(2) The potential therapeutic utility of Ephedra (3) Potential health risks associated with such uses
of Ephedra, particularly in sensitive individuals or in cases of intentional abuse for its stimulant properties.
Animal (14 W old ) treated with ephedrine (25 mg/kg) and caffeine (30 mg/kg), died few hours after 1st dosing. Hemorrhage (H) and
myofiber necrosis (N) in the left ventricle
H
H
N
N
H
Animal (14 W old ) treated with ephedrine (25 mg/kg) and caffeine (30 mg/kg), Died few hours after 1st dosing. Myofiber necrosis (N)
in the myocardium of the left ventricle
APOPTOTIC BODIES
MACROPHAGES
APOPTOTIC BODIES
APOPTOTIC BODIES
MACROPHAGES
Rat Heart Trimming Procedure
Julie F. Foley 3/12/05
The heart is sampled for:- Histopathology- Electron Microscopy- Toxicogenomic evaluation
Dorsal
Dorsal
On the dorsal aspect of the heart, place the blade below the pulmonary artery and cut a 3 mm transverse section.
Aorta
LA
V
Make first cut below this point. Make second cut3 mm below first cut.
The heart is now divided into 3 sections.• Top• Middle• Bottom
Top
Middle
Bottom
PA
Dorsal
Histology Section for Morphometric Analysis
For morphometry studies, it is important to standardize tissue collection. The transverse section of the heart is collected from the same region for each animal.
Remove the middle transverse section and placethe bottom cut surface down in a mega-cassettelined with an index card.
Use of a mega-cassette will prevent compression of the tissue. A piece of index card in the bottom of the cassette will minimize curling of the tissue and ensure a flat surface.
Fix the tissue overnight in 10% neutral buffered formalin. Remove the paper from the cassette before processing.
Cut surface down in mega-cassette.
Index card
LV
RV
Mega-cassette
Middle
Dorsal
Tissue for Electron Microscopy (EM)
Cut a 3 mm transverse section through the bottom of the heart. Representative cubes will be submitted for EM studies.
1 3
Representative cubes to submit for EM studies. 1) Right ventricular wall 2) Interventricular septum 3) Left ventricular wall
Discard or use for RNA studies.
LAA
orta
Top
Bottom
LVRV
PA
2
Longitudinal section bisecting the top of the heart.
Place cut side down of both halves of the top of the heart on an index card in a mega-cassette. Again, the mega-cassette will prevent compression of the tissue. The index card will minimize folding of the tissue. Remove the index card prior to the processing of the tissue by histology.
Aorta
LA
RA
Aor
ta
LVLV
RV
RV
RARA LA
V