Transcript Metabolism and Enzyme Kinetics in the Lung

Pulmonary Toxicology :
Disposition, Metabolism and
Enzyme Kinetics
Anthony J. Hickey, Ph.D., D.Sc.
School of Pharmacy, UNC-Chapel Hill, NC
• Introduction
• Lung Deposition
• Clearance Mechanisms
– Mucociliary Transport
– Cell Transport
– Absorption
•
•
•
•
Lung Cells
Enzyme Expression
Metabolism
Conclusion
Nasal
Passages
B
l
o
o
d
T-B Airways
Pulmonary
Parenchyma
Lymph
Nodes
G
I
T
r
a
c
t
Mucus
blanket
Cilia
Columnar
epithelial
cells
FRACTION CLEARED PER DAY (X103)
100
MICE
10
RATS
1
PEOPLE
DOGS AND GUINEA PIGS
0.1
100
200
DAYS AFTER INHALATION
300
1 µm polystyrene latex; 30 min; 60x
Thompson, 1992
Passive Diffusion
Facilitated Diffusion
Active Transport
CLEARANCE (min-1)
100
RAT
RABBIT
DOG
SHEEP
FETAL LAMB
MAN, AEROSOL
DOG, AEROSOL
10-1
10-2
10-3
10-4
10-5
101
102
103
104
105
MOLECULAR WEIGHT (daltons)
Effros and Mason, 1985
106
Aerosol
Throat
Cross section of two stages
Airflow
Pre-separator
1
Snapwell™
containing
epithelial cell
monolayer
2
3
4
5
Petri dish
Vacuum
6
To vacuum pump
Confluent monolayer of the
small airways epithelial cells
Airflow
Single Stage of the
Cascade Impactor
Showing Orifices
Transwell® Dish Containing
Epithelial Cell Monolayers
Petri Dish
Vacuum
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.
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)
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
• Introduction
• Lung Deposition
• Clearance Mechanisms
– Mucociliary Transport
– Cell Transport
– Absorption
• Lung Cells
• Enzyme
– Action
– Expression
– Distribution
• Conclusion
Cells of the Airway Epithelium
CELL
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
The rate of first-order kinetic reaction:
d [ S ] d [ P]


 k[ S ]
dt
dt
One-substrate mechanism:
E+S
k1
k2
ES
k3
E+P
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.
A Lineweaver-Burk plot
(based on Michaelis-Menten Equation)
Catalytic cycle of microsomal carboxylesterase
(left) and microsomal epoxide hydrolase (right),
two α/β-hydrolase fold enzymes.
Drug and Xenobiotic Metabolism
DRUG
SH
PHASE II
DRUG
Carboxyamide
OH
Glucuronic Acid
PHASE I
DRUG
Conjugation
NH3+
CO2-
SO4-
Cytochrome P450s
Glucuronosyltransferases
Monooxygenases
Sulfotransferases
Dehydrogenases
Acetyltransferases
Oxidases
Methyltransferases
Esterases
Glutathione S-Transferases
Glutathione
Functionalization
MDR1 (P-Glycoprotein)
EXCRETION
Courtesy: Matt Redinbo
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
Enzymatic Systems in the Respiratory Tract
• Phase II conjugating enzymes
–
–
–
–
Glutathione S-transferase (GST)
Sulfotransferase
N-acetyltransferase
methyltransferase
Summary of P-450 Isozymes Reported in
the Rat and Rabbit Nasal Cavities
Some P-450 Isozymes Reported in Lungs of
Various Species
Some P-450 Isozymes Reported in Lungs of
Various Species (Cont’d)
Isozyme
Comments
General pathways of xenobiotic biotransformation and their major subcellular location.
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
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
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
• Introduction
• Lung Deposition
• Clearance Mechanisms
– Mucociliary Transport
– Cell Transport
– Absorption
• Lung Cells
• Enzyme
– Action
– Expression
– Distribution
• Conclusion
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)
Structures of Some Acute Pulmonary Toxins
J.J. Fenton, Toxicology: A Case-Oriented Approach, CRC Press, Boca Raton, FL 2002.
Diesel Exhaust Particles
Solid carbon core (primary particle size of
10-80 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.
ROS Formation
DEP
Redox
Cycling
PAHs
Quinones
CYP1A1
ROS
ROS
NQO-1
Also from:
-activated macrophages
-recruited neutrophils
Hydroquinone
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.
Two-Electron Reduction of Menadione to a Hydroquinone,
and Production of Reactive Oxygen Species During its OneElectron 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.
Hierarchical Oxidative Stress Response
High
GSH/GSSG
Ratio
Low
GSH/GSSG
Ratio
Level of
Oxidative
Stress
Normal
Antioxidant
Defense
Inflammation
Cell or Tissue Response
Adapted from Xiao, et al. (2003). J Biol Chem. 278(50).
Toxicity
Scanning electron micrograph of an alveolar
macrophage
Macrophages as a host cell for infectious
microorganisms
Mycobacterium
tuberculosis
Toxoplasma
gondii
pH
NO
NO2NO3-
NH4+
H 2 O2
OH
O2
O2
SOD
Lysosomal
enzymes
GL
ST
LAM
NH4+
O2 NADP
NADPH
Conclusion
Particle deposition and distribution from the lungs is
mediated by a number of mechanisms
Conventional 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