Download Two-electron reduction of menadione to a hydroquinone, and

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
• 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.
Summary of Expression of Xenobiotic-Metabolizing Enzymes in
Human Lung —Phase I Enzymes
Enzymes
mRNA
Protein
Location
1A1
+++ (smokers)
+++ (smokers)
bronchial epithelial cells, capillary endothelium, alveolar epithelium
1A2
+/-
+/-
peripheral tissue
1B1
++
+/-
alveolar macrophage epithelial cells, bronchial epithelial cells
2A6
++
+/-
bronchial mucosa, epithelial cells
2A13
+
?
bronchial mucosa, alveolar epithelium
2B6/7
+++
+++
Clara cells, bronchial and peripheral tissue, epithelial cells
2C8/18
+
+/-
Serous cells of bronchial glands, bronchial and peripheral tissue
2D6
+/-
+/-
bronchial mucosa
2E1
+++
+++
bronchial, bronchiolar and alveolar epithelium, endothelial cells
2F1
+++
?
alveolar macrophage epithelial cells, endothelial cells
2J2
+
++
bronchial and vascular smooth muscle cell, vascular endothelium, alveolar macrophages
2S1
+
+
epithelial cells
3A4/5
+++
++
bronchial, bronchiolar and alveolar epithelium, alveolar endothelium,
alveolar macrophages
3A43
+
?
?
4B1
+
?
bronchoalveolar macrophages
EHs
++
++
bronchial epithelial cells
Phase I
CYPs
FMOs
++
+
?
- weak negative evidence, +/- conflicting evidence, + weak positive evidence, ++ moderate positive evidence, +++ strong evidence, ? no report
Zhang, J. Y.; Fen Wang, Y.; Prakash, C., Current Drug Metabolism 2006, 7, 939-948.
Summary of Expression of Xenobiotic-Metabolizing
Enzymes in Human Lung —Phase II Enzymes
Enzymes
mRNA
Protein
Location
+/-
+/-
lung tissue
A1
++
+
bronchial and bronchiolar epithelium
A2
++
+
bronchial and bronchiolar epithelium
M1
++
+
lung tissue
M2
++
+
epithelium of the terminal airways
M3
++
+
ciliated airway epithelium and smooth muscle
P1
++
+
bronchial and bronchiolar epithelium
NATs
+
+
bronchial epithelial cells, alveolar lining cells
1A1
+
+
bronchial epithelial cells
2B1b
+
+
bronchial epithelial cells
Phase II
UGTs
GST
SULTs
- weak negative evidence, +/- conflicting evidence, + weak positive evidence, ++ moderate positive evidence, +++ strong evidence, ? no report
Zhang, J. Y.; Fen Wang, Y.; Prakash, C., Current Drug Metabolism 2006, 7, 939-948.
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
Comparative Metabolism of Trichloroethylene (TCE) in
Mouse Hepatocytes and Lung Clara Cells
Mouse Hepatocytes
Cl
Cl
Cl
CYP2E1
H
CCl3CHO
90%
Chloral
Trichloroethylene
10%
CCl3CH 2OH
CCl3CH 2OGluc
Trichloroethanol
Trichloroethanol
glucuronide
CCl3COOH
Trichloroacetic acid
Mouse Lung Clara Cells
Cl
Cl
Cl
H
CYP2E1
CCl3CHO
CCl3CH 2OH
Excess Chloral
Trichloroethylene
CCl3COOH
Clara Cell Damage
Green, T., Environ Health Perspect 2000, 108 Suppl 2, 261-4.
CCl3CH 2OGluc
The Relevance of Clara Cell in Assessing Human
Risks of Toxicity: Noticing Specie Differences

Different expression levels of CYP2E1 between species:
mouse > rat > human
TCE metabolism is 600-fold less in human lung than in mouse lung,
due to lack of expression of CYP2E1 in any cell type.
Number and morphology differences of Clara Cells
between species


Implication of involvement of alveolar type II cells
A large number of mouse lung tumors express alveolar type II
surfactant apoprotein.
Green, T., Environ Health Perspect 2000, 108 Suppl 2, 261-4.
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