pulmonary toxicology : disposition, metabolism and enzyme kinetics anthony j. hickey, ph.d., d.sc....
TRANSCRIPT
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Pulmonary Toxicology : Disposition, Metabolism and
Enzyme Kinetics
Anthony J. Hickey, Ph.D., D.Sc.
School of Pharmacy, UNC-Chapel Hill, NC
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• Introduction
• Lung Deposition
• Clearance Mechanisms– Mucociliary Transport– Cell Transport– Absorption
• Lung Cells
• Enzyme Expression
• Metabolism
• Conclusion
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NasalPassages
T-B Airways
PulmonaryParenchyma
LymphNodes
Blood
GI
Tract
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MucusblanketCilia
Columnarepithelial cells
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MICE
RATS
PEOPLE
DOGS AND GUINEA PIGS
100 200 300
100
10
1
0.1
DAYS AFTER INHALATION
FRAC
TIO
N CL
EARE
D PE
R DA
Y (X
103 )
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1 µm polystyrene latex; 30 min; 60x
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Thompson, 1992
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Passive Diffusion
Facilitated Diffusion
Active Transport
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MOLECULAR WEIGHT (daltons)
CLEA
RANC
E (m
in-1)
RAT
RABBIT
DOG
SHEEP
FETAL LAMB
MAN, AEROSOL
DOG, AEROSOL
100
10-1
10-2
10-3
10-4
10-5
101 102 103 104 105 106
Effros and Mason, 1985
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Aerosol
To vacuum pump
Cross section of two stages
Throat
Pre-separator
1
2
3
4
5
6
Airflow
Vacuum
Snapwell™ containing epithelial cell monolayer
Petri dish
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Confluent monolayer of the small airways epithelial cells
Vacuum
Airflow
Single Stage of the Cascade ImpactorShowing Orifices
Petri Dish
Transwell® Dish Containing Epithelial Cell Monolayers
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Relationship between clearance from the lungs and molecular weight of FITC-dextrans
Arrows indicate the positions of 4.4, 9.5, 21.2, 38.9 and 71.2 kD markers. Dotted lines represent the range of the data obtained from the different species.
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FITC-Dextran Average MW (kD) 9.5 21.2 38.9 71.2
Relative Permeability:
Papp(4.4kD)/Papp(x kD), (n=3)
3.0 2.9 4.9 12.9
Relative In-vivo Clearance:
Cl(4.4kD)/Cl(x kD)4.4 6.2 6.6 20
Comparison of relative permeability coefficients determined using in vitro model and relative in vivo clearance from the lungs for
FITC-dextrans (4.4:9.5kD; 4.4:21.2kD; 4.4:38.9kD; and 4.4:71.2kD)
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• Introduction
• Lung Deposition
• Clearance Mechanisms
– Mucociliary Transport
– Cell Transport
– Absorption
• Lung Cells
• Enzyme
– Action
– Expression
– Distribution
• Conclusion
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Cells of the Airway EpitheliumCELL PUTATIVE FUNCTION
Ciliated columnar Mucus movement
Mucus (goblet) Mucus secretion
Serous Periciliary fluid
Clara (nonciliated epithelial) Surfactant production, xenobiotic metabolism
Brush Transitional form of ciliated epithelial cell
Basal Progenitor for ciliated epithelial cell and goblet cell
Intermediate Transitional cell in differentiation of basal cell
Neuroendocrine Chemoreceptor, paracrine function
Alveolar Type I Alveolar gas exchange
Alveolar Type II Surfactant secretion, differentiation to type I cell
Alveolar Macrophages Pulmonary defense
Mast Immunoregulation
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E + S ES E + Pk1
k2
k3
][][][
Skdt
Pd
dt
Sd
The rate of first-order kinetic reaction:
One-substrate mechanism:
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A. Dependence of initial rate of reactant concentration for a simple first- or second-order chemical reaction.
B. Dependence of initial rate of substrate concentration for a typical enzyme-catalyzed reaction.
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A Lineweaver-Burk plot(based on Michaelis-Menten Equation)
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Catalytic cycle of microsomal carboxylesterase (left) and microsomal epoxide hydrolase (right), two α/β-hydrolase fold enzymes.
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Drug and Xenobiotic Metabolism
DRUG DRUG
OH SH
NH3+ CO2
-
DRUGG
luc
uro
nic
A
cid
SO4- G
luta
thio
ne
Ca
rbo
xy
am
ide
PHASE I PHASE II
Functionalization Conjugation
EXCRETION
MDR1 (P-Glycoprotein)
Cytochrome P450s
Monooxygenases
Dehydrogenases
Oxidases
Esterases
Glucuronosyltransferases
Sulfotransferases
Acetyltransferases
Methyltransferases
Glutathione S-Transferases
Courtesy: Matt Redinbo
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Enzymatic Systems in the Respiratory Tract
• Phase I – CYP-450s– Flavin containing mono-oxygenases (FMA)– Monoamine oxidase (MAO)– Aldehyde dehydrogenase– NADPH cP450 reductase– Esterases– Epoxide hydrolase
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Enzymatic Systems in the Respiratory Tract
• Phase II conjugating enzymes– Glutathione S-transferase (GST)– Sulfotransferase– N-acetyltransferase– methyltransferase
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Summary of P-450 Isozymes Reported in the Rat and Rabbit Nasal Cavities
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Some P-450 Isozymes Reported in Lungs of Various Species
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Some P-450 Isozymes Reported in Lungs of Various Species (Cont’d)
Isozyme Comments
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General pathways of xenobiotic biotransformation and their major subcellular location.
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Distribution of Enzymes
• Upper respiratory tract– Olfactory epithelium:
• CYP450 & NADPH
• CYP450 levels < liver, but activities >> than liver
• Epoxide hydrolase, carboxylesterase, aldehyde dehydrogenase activity > respiratory
• Phase II enzymes: GST, glucoronyl transferases, sulfotransferases
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Distribution of Enzymes• Lower respiratory tract• Tracheobronchial region
– CYP450 throughout
– FMO absent in larynx and trachea
• Bronchiolar region– Clara cells:
• CYP450 isozymes
• NADPH cP450 reductase
• FMO, GST, UDP-GT, and epoxide hydrolase
– Type II pneumocytes • CYP450 isozymes
• NADPH cP450 reductase
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Distribution of Enzymes
• Alveolar Macrophages:– No CYP450
• Type I cells– No metabolic activity– Susceptible to toxicity e.g. butylated
hydroxytoluene is severely toxic to Type I cells
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• Introduction
• Lung Deposition
• Clearance Mechanisms
– Mucociliary Transport
– Cell Transport
– Absorption
• Lung Cells
• Enzyme
– Action
– Expression
– Distribution
• Conclusion
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Pulmonary Enzyme Systems
• CYP450 mono-oxygenase– Metabolism of endogenous FA’s, steroids, and lipid
soluble xenobiotics
– Note: some metabolism leads to bioactivity or carcinogens (e.g. benzo[a]pyrene)
• NADPH Cytochrome P450 reductase– Identical to hepatic enzyme
– Activates toxicity of paraquat and nitrofurantion (reduction of nitro grp free radical regenerates parent drug and superoxide anion lipid peroxidation and depletion of cellular NADPH)
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Structures of Some Acute Pulmonary Toxins
J.J. Fenton, Toxicology: A Case-Oriented Approach, CRC Press, Boca Raton, FL 2002.
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Diesel Exhaust Particles
Solid carbon core (primary particle size of10-80 nm, agglomerates of 50-1000 nm).
Adsorbed hydrocarbons.
Liquid condensed hydrocarbon particles.
Sulfates, nitrates, metals, or trace elements.
Adapted from Marano, et al. (2002). Cell Biol Toxicol. 18(5): 315-320.
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ROS FormationDEP
QuinonesRedoxCycling
ROS
PAHs
ROS
Hydroquinone
NQO-1
CYP1A1
Also from:-activated macrophages-recruited neutrophils
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Role of epoxide hydrolase in the inactivation of benzo[a]pyrene 4,5-oxide and in the conversion of benzo[a]pyrene to its tumorigenic diolepoxide.
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Two-Electron Reduction of Menadione to a Hydroquinone, and Production of Reactive Oxygen Species During its One-
Electron Reduction to a Emiquinone Radical
Casarett and Doull’s Toxicology: The Basic Science of Poisons,C.D. Klaassen Ed., 6th Ed. McGraw-Hill, New York, NY 2001.
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Normal AntioxidantDefense
Inflammation Toxicity
HighGSH/GSSG
Ratio
LowGSH/GSSG
Ratio
Level ofOxidative
Stress
Adapted from Xiao, et al. (2003). J Biol Chem. 278(50).
Hierarchical Oxidative Stress Response
Cell or Tissue Response
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Scanning electron micrograph of an alveolar macrophage
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Macrophages as a host cell for infectious microorganisms
Mycobacterium tuberculosis
Toxoplasma gondii
NONO2
-
NO3-
pHH2O2
OHO2
O2
SODO2
-
NADPLysosomalenzymes NH4
+
NADPHNH4+
GLSTLAM
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ConclusionParticle deposition and distribution from the lungs is
mediated by a number of mechanismsConventional enzyme kinetic analysis may be used to
characterize activity in lung tissue (fluids or cells).There are a number of cell types throughout the respiratory
tract exhibiting differential enzyme expression and activity.
Local metabolism of xenobiotics may result in toxicity (metabolism of drugs may result in efficacy or inactivation).
Pathogens act, in part, by suppressing metabolism