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Effect of cigarette smoke on redox regulation in chronic obstructive pulmonary disease

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Effect of cigarette smoke on redox regulation in chronic obstructive pulmonary disease

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EFFECT OF CIGARETTE SMOKE ON REDOX REGULATION IN
CHRONIC OBSTRUCTIVE PULMONARY DISEASE
by
Amit Agarwal

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCE)

August 2010

Copyright 2010 Amit Agarwal
ii

DEDICATION

This thesis is dedicated to my mentor of life Madhu Mhasawde,
who helped me realize my potential and be the person I am today.
It is all because of you.
Thank you for all the guidance, support and love which you gave
me during the good times and bad.
I feel blessed to have met you and hope that you will keep
showering your tender love from the skies.
Miss you!

iii

ACKNOWLEDGMENTS

I would like to express my heartfelt appreciation to the committee reviewing my
thesis, Dr. Enrique Cadenas, Dr. Wei-Chiang Shen, and Dr. Curtis Okamoto.
Thank you for giving me an opportunity to be a part of research in the great
School of Pharmacy. Special thanks to Dr. Cadenas for having faith in me and
supporting me throughout my Masters.
I would also like to appreciate the guidance provided by Li Peng Yap, Jerome
Garcia and Ryan Hamilton. It is from you people that I have learnt everything I
know. Thank you to lab members Harsh, Chen, and Fei for all the support.
The most special thanks to my parents Rajendra and Rita Agarwal and my lovely
little sister Neha, for their continuous support and love all my life and last but
never the least, thank you GOD.

iv

TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT ix
HYPOTHESIS 1
INTRODUCTION
Chronic obstructive pulmonary disease (COPD)
Free radicals and electrophiles in cigarette
The cellular redox environment: an indicator of cellular health
Protein thiol modification
Role of mitochondrial dysfunction in COPD
Mitochondrion driven apoptosis in COPD
The pentose phosphate pathway

1
1
2
2
4
5
8
9
METHODS
Preparation of cigarette smoke extract
Cell culture and treatment
Exposure of mice to cigarette smoke
Isolation of lung mitochondria
Cell viability assay
Western blots analyses
Seahorse XF-24 metobolic flux analysis
Enzyme activity assays
Quantitation of pyridine nucleotides in RLE 6TN cells and lung mitochondria
Quantitation of thiols in RLE 6TN cells and lung mitochondria
11
11
13
14
15
16
16
17
17
18
19

v

RESULTS
Effects of CSE, acrolein, and nitric oxide on cell viability in RLE6TN cells
Effect of CSE on mitochondrial respiration and glycolysis in RLE6TN cells
Effect of CSE treatment on NADPH/NADP
+
and NADH/NAD
+
ratios
Effect of CS treatment on NADPH/NADP
+
and NADH/NAD
+
ratios in male
A/J mice lung mitochondria
Effect of CSE treatment on GSH/GSSG ratios and GSNO levels
Effect of CS treatment on GSH/GSSG ratio and GSNO in male A/J mice lung
mitochondria
Glutathionylation on exposure to CSE
Glucose 6 Phosphate dehydrogenase and Glyceraldehyde 3 Phosphate
dehydrogenase enzyme activity

21
21
24
27

30
31

34
35

36
DISCUSSION 40
REFERENCES 43
ALPHABETIZED BIBLIOGRAPHY 50

vi

LIST OF TABLES

Table I. 2 weeks cigarette smoke exposed lung mitochondria shows an
increase in NADPH/NADP+ and NADH/NAD+ ratios when
energized using glutamate/malate. When not energized no
significant decrease was found in the NADPH/NADP+ with
a slight decrease in the NADH/NAD+.

30
Table II. 2 weeks cigarette smoke exposed lung mitochondria shows
an increase in GSH/GSSG ratio and GSNO levels when
energized using glutamate/malate. The ratio increased
even in the absence of G/M.

34

vii

LIST OF FIGURES
Figure 1. Redox modulation of thiols 4
Figure 2. Cell death as a function of Nernst potential 7
Figure 3. The pentose phosphate pathway and its neuroprotective role
against Nitrosative stress

10
Figure 4. Absorption spectra of CSE in serum free F12 12
Figure 5. Dose dependent effects of acrolein, nitric oxide, and CSE
(short-term and long-term) on cell viability measured by
MTT assay in RLE6TN cells.
(A) RLE6TN cell viability with 24-h acrolein exposure.
(B) RLE6TN cell viability with 24-h nitric oxide exposure.
(C) RLE6TN cell viability with 4 h CSE exposure.
(D) RLE6TN cell viability with 24 h CSE exposure.

21
22
22
23
Figure 6. CSE treatment leads to impairment of oxidative
phosphorylation and a shift towards glycolysis.
A) OCR from RLE6TN cells treated with 0.5 and 1.75%
CSE was compared with OCR from untreated cells.
B) The OCR/ECAR ratio showed a shift towards glycolysis
on 1.75% CSE treatment in RLE6TN cells.

24
25
Figure 7. A) Short-term (4h) CSE treatments lead to an increase
in the NADPH/NADP
+
,
B) with no effect on NADH/NAD
+
ratio.
C)Prolonged (24h) CSE treatment leads to a decrease in
NADPH/NADP
+

D) and NADH/NAD
+
ratio in RLE6TN cells.

27
28

28
29
Figure 8. A) Short term (4h) CSE treatment leads to an
increase in the GSH/GSSG ratio
B) Long term (24h) CSE treatment leads to a
decrease in the GSH/GSSG ratio in RLE-6TN cells.
C) GSNO levels after 4h CSE exposure.
D) GSNO levels after 24h CSE exposure.

31

32
32
33
Figure 9. Western blot from 4h CSE exposed RLE 6TN cell extract.

35
Figure 10. A). Glucose 6 Phosphate dehydrogenase enzyme
activity from RLE-6TN cells exposed to CSE for 4 h.

36

viii

B). Glucose 6 phosphate dehydrogenase enzyme activity from
2 weeks smoke exposed male A/J mice lung cytosol.

37
Figure 11. A) Glyceraldehyde 3 Phosphate dehydrogenase
enzyme activity form 4 h CSE exposed RLE-6TN cell extract.
B). Glyceraldehyde 3 Phosphate dehydrogenase enzyme
activity form 2 week smoke exposed male A/J mice lung cytosol

38

38

ix

ABSTRACT
Chronic obstructive pulmonary disease (COPD) is a persistent obstruction of
airways in the lungs due to the narrowing of the passage ways. Cigarette smoking is
responsible for approximately 80-90% of COPD-related deaths. Cigarette smoke contains
over 4000 chemicals, including carcinogens, strong electrophiles and other toxins. The
current study was undertaken to determine the role of cigarette smoke in the development
of mitochondrial dysfunction due to impairment of the energy-redox axis. Rat lung
epithelial cell cultures (RLE-6TN cell line) and male A/J mice were used to study the
effects of cigarette smoke extract (CSE) or cigarette smoke (CS), respectively. The
effects of individual constituents of CSE like acrolein and nitric oxide on cell viability
and redox status of the cell was also assessed. We have adapted a method to prepare
cigarette smoke extract in our laboratory by smoking 4 cigarettes in 20 ml of serum free
F-12 media. Our data indicate that treatment of RLE-6TN cells with doses less than 2.5%
CSE for 4 h, and whole body smoke exposure for 2 weeks in A/J mice lead to an increase
in the NADPH/NADP
+
ratio with no change in the NADH/NAD
+
ratio as measured by
HPLC. At 24 h, a decrease in both NADPH/NADP
+
and NADH/NAD
+
ratios, indicate of
an initial compensatory response to cigarette smoke. This increase in NADPH was found
to be because of the increased activity of the pentose phosphate pathway. The other redox
couple of GSH/GSSG was also shown to increase.

1

HYPOTHESIS
Cigarette smoke alters the redox status of the cells by modulating the pools of
redox couples like NADPH/NADP
+
and GSH/GSSG leading to mitochondrial
dysfunction and apoptosis, which is inherent in COPD pathology. This in effect
modulates the activity of metabolic enzymes that may be the target of protein S-
glutathionylation caused by increased mitochondrial oxidants and disruption of redox
signaling and control.

INTRODUCTION
Chronic obstructive pulmonary disease (COPD)
COPD is one of the leading causes of mortality and morbidity worldwide. COPD
is estimated to be the fourth leading cause of deaths worldwide by 2030
1
. In 2007, 12
million people were identified with COPD and another 12 million were considered to be
undiagnosed
2,3
. The global initiative for chronic obstructive lung disease (GOLD) defines
COPD as ―a disease state characterized by airflow limitation that is not fully reversible.
The airflow limitation is usually both progressive and associated with an abnormal
inflammatory response of the lungs to noxious particles or gases
4
. COPD is characterized
by development of broadly two kinds of pathologies, chronic bronchitis or emphysema.
While chronic bronchitis involves production of excessive cough due to underlying
inflammation in the goblet cell that is the mucus producing cell, leading to fibrosis
5
,
emphysema is characterized by a destruction of alveolar bronchioles and enlargement of
air spaces without development of fibrosis. Oxidant/antioxidant imbalance is believed to

2

be one of the major causes in the development of COPD as it can lead to inflammation
which potentiates proteolytic damage, induces cell death and inhibits cell repair
6,7
.
Free radicals and electrophiles in cigarette
CS is an extremely rich source of oxidants and is estimated to contain
approximately 10
17
radicals per gram in the tar phase and 10
15
radicals in the gaseous
phase.
8
Seminal work identified the principle radical in tar phase as a semiquinone that
is capable of reducing molecular oxygen to O
2
.–
, eventually leading to H
2
O
2
9
. Divalent
metal ions that are potentially chelated by tar can in turn produce the highly reactive
hydroxyl radical (HO
.
) through the Fenton reaction (Me
n
+ H
2
O
2
→ Me
n+1
+ HO
–
+ HO
.
).
It has been estimated that the amount of ROS released from a cigarette was ~384 nmol in
research grade cigarettes to 414 nmol in commercially-available cigarettes without filters
and 288 nmol in cigarette with filters
10
. CS contains as much as 500 ppm NO

and this NO
can be oxidized to form the more reactive NO
2
radical
8,11
. ONOO
–
can also be formed
under these conditions
8,12
. Although there are approximately 4700 chemicals found in
CS, the biologically important radicals contained in CS are the O
2
.-
, HO
.
,
.
NO, NO
2
and
oxidants H
2
O
2
and ONOO
-11
. End-product specific chemical post-translational
modifications of proteins, such as protein carbonylation and tyrosine nitration, are an
indicator for specific ROS and RNS damage.

The cellular redox environment: an indicator of cellular health
Glutathione (GSH) is a small hydrophilic molecule that is synthesized in the
cytosol from glycine, glutamate, and cysteine in a two-step process by the enzymes γ-

3

glutamylcysteine synthetase and GSH synthase
13
. As the most abundant non-protein
thiol
14
, GSH plays a central role as an antioxidant. The oxidation product of GSH, GSSG,
is reduced back to GSH by glutathione reductase, a NADPH-dependent enzyme
ubiquitously distributed throughout the cell and tissues. Under non-oxidative or
nitrosative stress conditions, the concentration of GSSG is negligible at 1/100
th
of the
total GSH pool
15
. Measurement of GSH and GSSG levels has been used to define the
redox environment of the cell. The redox state of a redox couple can be defined by the
half-cell reduction potential; the redox potential can be calculated using the Nernst
equation (E
hc
= E
o
– RT/nF ln Q, where R is the gas constant, T is the temperature in
Kelvins, and F is the faraday constant, n is the number of electrons exchanged and Q is
the mass action exchanged). Thus, for GSH/GSSG, at 25
o
C, pH7.0, the redox potential
for GSH is defined as E = –240 –(59.1/2) log([GSSG]/[GSH]
2
)
15
. This is extremely
useful, as this allows comparison of the reducing force available from the GSSG/2GSH
couple with respect to other redox couples. As the concentration of GSH far exceeds any
other redox couple (~100-10,000 greater)
16
, the redox status of the cell can be assessed by
assessing the redox potential for GSH/GSSG. The redox status of the cell is associated
with a cell’s progression through its life cycle: as the redox environment becomes
increasingly oxidized, the cell progresses from proliferation to differentiation to apoptosis
and necrosis
15
.
Our laboratory has previously shown that at low concentrations of H
2
O
2,
where
the redox status is less oxidized, cells undergo apoptosis; however, at higher
concentrations of H
2
O
2
, the cellular redox status becomes more oxidized, shifting the

4

mode of cell death from apoptosis to necrosis
17
. Additionally, exposure of fibroblast cells
to CSE resulted in a decrease of GSH levels without the concomitant increases in
GSSG
18
. Depletion of GSH in RLE-6TN, an alveolar epithelial cell line, increases
sensitivity towards H
2
O
2
and hyperoxia
19
. Patients with reversible air-flow limitation in
COPD showed lower levels of GSH oxidation and protein oxidation than patients with
COPD with no reversibility
20
. Taken together, in vitro data strongly supports the concept
that CS exposure results in oxidation of the redox cellular environment and alterations in
cellular GSH/GSSG induced cell death. Severity of symptoms and reversibility also
seem to correlate with the extent of oxidative challenge.

Protein thiol modifications: a redox sensitive switch
The sulfhydryl groups in the vast
majority of protein cysteine residues are
redox sensitive, that is, susceptible to redox
changes in the cellular environment21
21,22
.
The GSH/GSSG redox couple can regulate
protein function through the reversible
formation of mixed disulfides that can occur
mainly through three mechanisms. (i) thiol
disulfide exchange between protein
sulfhydryl groups and GSSG, (ii) GSH
reduction of protein sulfenic acids, and (iii)

5

the nucleophilic attack of thiolate anion on the S-NO bond of GSNO
15,23
(Fig. 1). The
formation of mixed disulfides reflects the redox status of the cell
15
. Unlike S-
nitrosylation, protein-mixed disulfides are specifically reduced by glutaredoxins, which
catalyze the reversible transfer of the glutathionyl moiety via an enzyme-glutathione-
mixed disulfide intermediate. The reversible formation of mixed disulfides has been
suggested as a mechanism that protects critical sulfhydryls from irreversible damage and
also has significance with regard to redox regulation of signal transduction
23
. In intact rat
liver mitochondria, the proportions of exposed and reactive thiols are 5-fold higher than
that of GSH suggesting that the relationship between exposed protein thiols and GSH
plays a key role in mitochondrial thiol metabolism
24
.

Role of mitochondrial dysfunction in COPD
There are host of evidences suggesting the role oxidative stress and mitochondrial
dysfunction in a spectrum of diseases. The role of mitochondria in cellular pathology is
reflected in its role as the powerhouses of the cell through the generation of ATP during
oxidative phosphorylation, as well as the generation of reactive oxygen and nitrogen
species and permeability transition.
 Mitochondria are the major cellular source of oxidants – Following initial reports
by Chance & Boveris on the production of H
2
O
2
by intact mitochondria
25
,
subsequent work established that O
2
.–
was the stoichiometric precursor of
mitochondrial H
2
O
2
and that it was primarily generated during ubisemiquinone
autoxidation
26,27,28
and secondarily, by NADH-dehydrogenase activity
29
. The

6

physiological generation of O
2
.-
occurs due to the oxidation of the components of
the electron transport chain (complex I and III)
30
. O
2
.–
formed during electron
transfer at the inner membrane, can also be vectorially released into the
intermembrane space
31
where it is converted to H
2
O
2
by the Cu,Zn-superoxide
dismutase present in this compartment. The mitochondrial production of H
2
O
2
is
regulated by the mitochondrial metabolic state and intra-mitochondrial steady-
state concentration of
.
NO.
 Regulation of mitochondrial functions by nitric oxide – Cytochrome oxidase
(COX, complex IV) is the terminal component of the electron transport chain and
catalyses the oxidation of cytochrome c and the reduction of O
2
to water in a
process that is linked to proton pumping out of the matrix.
.
NO binds to the
binuclear center
32
of COX and inhibits its activity in a reversible and O
2

competitive manner
33
. A new concept of the regulation of cellular respiration was
advanced that retains the classical concept that energy demands drive respiration
but kinetic control of both respiration and energy supply is dependent upon the
availability of ADP to F
1
-ATPase and O
2
and
.
NO to COX
34
. Hence, in addition
to regulating COX activity, NO brings about a metabolic hypoxia where due to
increases in NO, such as during inflammation or degenerative disease, available
O
2
cannot be adequately used
35
. At physiological concentration of
.
NO, inhibition
of mitochondrial respiration occurs with a concomitant increase in O
2
.–
and
ONOO
–
formation
36
. CS contains high amounts of
.
NO
8
and lymphocytes
collected from COPD patients had impaired complex III and IV activity
37
.

7

Therefore, it is reasonable to postulate that CS contains components (e.g.,
.
NO)
capable of regulating mitochondrial complex activities and increase oxidant
production through the mitochondrial electron transport chain.
 Chemical modification of redox sensitive mitochondrial proteins: Many
mitochondrial proteins such as Mn-SOD
38
, complex I
39
and aconitase
40
have been
shown to be chemically modified during oxidative and/or nitrosative stress. In
vitro studies using isolated mitochondria showed that complex I can undergo
post-translation
modifications such as
nitration by ONOO
-

and S-
glutathionylation,
resulting in loss of
complex I activity
41,42
S-glutathionylation
can also lead to modulation of aconitase activity under oxidative and nitrosative
stress
40
. Although no specific modification of mitochondrial proteins have been
observed in COPD, we do know that activities of the various complexes of the
mitochondrial respiratory chain are impaired. Considering the plethora of
mitochondrial proteins that are susceptible to chemical protein post-translational
modifications, it might be reasonable to suggest that CS-induced oxidative and
nitrosative stress could lead to mitochondrial protein modifications. Impairment

8

of mitochondrial protein function has specific consequences which include
increase ROS and RNS production, energy deficit, collapse of mitochondrial
membrane potential that leads to loss of ATP production and mitochondrial
permeability transition, an initiation of the commitment phase of mitochondrion-
driven apoptosis.
At the mitochondrial level, a multitude of pathways could potentially converge, the
information integrated and subsequently the decision to live or die is made. This is
extremely important as it highlights the central role of mitochondria as a propagator of
oxidants in COPD as well as an executioner of cell death. Permanent damage to these
organelles could lead to continuous cellular dysfunction, decline in cellular energy,
increased oxidant production and death, even in the event of smoking cessation.

Mitochondrion driven apoptosis in COPD
Mitochondria contain sensors of oxidative stress, namely in the form of
sulfhydryl groups of redox-sensitive proteins such as adenine nucleotide translocase
(ANT). The normal function of this protein is to exchange ADP and ATP across the inner
membrane; however, when sulfhydryl groups on ANT are modified by oxidative stress or
nitrosative stress (S-nitrosylation), ATP binding to ANT is inhibited as well as the Ca
2+

trigger site. As a consequence, ANT becomes a nonspecific structural pore within the
inner mitochondrial membrane and becomes part of the mitochondrial permeability
transition pore, PTP
43
. The most important consequences of the opening of the PTP are
uncoupling of mitochondrial respiration, mitochondrial swelling, and rupture of the outer

9

mitochondrial membrane with the release of apoptotic factors such as cytochrome c and
the activation of apoptotic pathways
44
. Several lines of evidence support mitochondrion-
driven apoptosis of epithelia in COPD. The presentation of apoptotic cells in the lung
of patients with COPD is well documented
45
.
Apoptotic cells in lung parenchyma from patients suffering from COPD
consisted of endothelial cells, alveolar, and bronchial epithelial cells
51
. Mitochondria
activated caspase-9
46
, in infant lungs of monkeys that were exposed to tobacco smoke in
uterus
47
. The same study also showed a parallel increase in expression of Bcl-2
and Bcl-x
L
antiapoptotic proteins that regulate cytochrome c release from mitochondria
through non-descript pores
48
. Lung epithelial cells (pretreated with staurosporin, a
trigger of apoptosis that does not affect mitochondrial respiration) exposed to CS
switched from apoptosis to necrosis
49
. Exposure of bronchial epithelial cells to CSE
resulted in shift of cell death from apoptosis to necrosis
50
. It may be surmised that
mitochondrion-driven apoptosis occurs in COPD and lung cells have a ―switch‖ point,
which can be defined by the Nernst potential, where there is a complete collapse of
cellular ATP levels and cells die by necrosis (Fig.2).

The pentose phosphate pathway
The pentose phosphate pathway in the cytosol is responsible for 10-20% of the
glucose metabolism. It functions to produce NADPH which is utilized to maintain the
redox status of the cell to a more reduced form by regenerating glutathione and in the

10

process protecting the
cell from oxidative
damage. This reducing
power is also utilized in
the biosynthesis of fatty
acids to reduce the
double bonds of the
intermediates
56
. Another
important function of
this pathway is to
produce ribose-5-
phosphate which is used in the synthesis of nucleotides and nucleic acids
52
. The pathway
is particularly upregulated under conditions of oxidative damage (to cope with the
oxidized environment), hypoxia (particularly in ischemia through glucose-6-phosphate
dehydrogenase enzyme activation) and cancer (synthesis of nucleic acids in malignant
cells)
52
. Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose
phosphate pathway and is known to be activated by nitrosative stress. Bolaños and group
have shown that nitric oxide activates G6PD to promote neuronal survival by balancing
the energy metabolism between the PPP and glycolytic pathways
53,54
. ONOO
-
has been
shown to trigger rapid stimulation of PPP and accumulation of NADPH
55
. Since nitric
oxide is one of the constituents of cigarette smoke, it can be expected to have similar
effects on the energy metabolism of the cells.

11

METHODS
Preparation of cigarette smoke extract
Aqueous cigarette smoke extract was prepared using research grade cigarette
(3R4F) from the Kentucky Tobacco Research and Development Center at the University
of Kentucky (Lexington, KY) containing total particulate matter (11.0 mg/cigarette), tar
(9.4 mg/cigarette), and nicotine (0.73 mg/cigarette)
57
. The cigarettes were stored in a
dessicator at least 48 h prior to use with a solution of glycerin/water (mixed in a ratio
0.76/0.26) to establish a relative humidity (RH) of 60%. The extract was prepared by
bubbling smoke from 4 cigarettes into 20 ml of serum free F-12 media in a conical flask
fitted with an inward and outward port at the rate of 1 cigarette per 30 s till 0.5 cm above
the filter using a modification of the method previously described by Carp and Janoff
58
.
The smoke was drawn using a Welch
®
DRYFAST
®
Collegiate Model 2014 vacuum
pump into the media. To ensure consistency with the preparation of the extract, the
smoke was allowed to settle on the media for 1 min between bubbling of smoke from 2
cigarettes. After the smoke from 4 cigarettes was bubbled into the media in the fumehood
another 4 min were allowed for the smoke to completely settle on the media. Once the
smoking was complete the extract was filtered using a 0.22 µm Steriflip vacuum filtration
system from Millipore (Billerica, MA) to sterilize and eliminate the particulate matter.
The extract was used in no more than 30 min after preparation. The spectrophotometric
analysis of the smoke extract was done using an Agilent 8453 UV-Visible
Spectrophotometer and a consistent peak was obtained at 361 nm. There appears to be a
high level of discrepancy between laboratories in the method of preparing and analyzing

12

the cigarette smoke extract
59, 63
. This discrepancy arises from the lack of a standard, well-
established procedure for the preparation of cigarette smoke extract. MacNee et al use a
cigarette smoke condensate for these experiments where they smoke around 3 cigarettes
in 3 ml of PBS to get a concentrated extract and then dilute it as per their
requirement
60,61,62
. Yao et al use a cigarette smoke extract prepared by smoking 1
cigarette in 10 ml of media at the rate of around 2 min/cigarette and then uses this extract
as 100% CSE
63,64
.

Fig 4. Absorption spectra of CSE in serum free F12

-0.2
0
0.2
0.4
0.6
0.8
1
1.2
190
222
254
286
318
350
382
414
446
478
510
542
574
606
638
670
702
734
766
798
830
862
894
926
958
990
1022
1054
1086
Absorbance
Wavelength
1 cigarette
2 cigarettes
3 cigarettes
4 cigarettes

13

The problem with this method of preparation of CSE is that because the smoke is
bubbled directly into an aqueous solution, there is loss of the constituents in the vapor
phase, which are insoluble in aqueous solvents, such as quinones, and the filtering takes
out the particulate matter. Some groups have tried to negate this effect by directly
exposing the cells to cigarette smoke. In this case the flask is turned upside down and the
vapors are made to pass over the cells
65
. The major issue with this method is the
consistency in the amount of cigarette smoke being exposed to cells. Also it is very
difficult to maintain consistency in the preparation of CSE because it can vary with a
number of factors such as length of cigarette smoked, rate at which the cigarette is
smoked, the pH of the CSE, etc. Thus a spectrophotometric analysis of the smoke extract
appears to be a requirement to standardize each cigarette smoke extract.
While most of the groups fail to mention the reference wavelength that was used
to standardize the CSE, there is not consensus with those who do. A few groups have
standardized the CSE with a peak at 320 nm
59,66
, but in our case a peak at 361 nm was
obtained as shown in the spectra using various solutions like PBS, HBSS, and F12 . We
have used this wavelength to standardize the CSE for all of our experiments. The pH of
the CSE was 7.0. The spectra shows that after smoking 4 cigarettes the extract becomes
saturated with no increase in optical density.

Cell culture and treatment
Rat lung epithelial-T-antigen negative (RLE6TN) cells were obtained from
(ATCC, Manassas, VA). The cells were maintained in Ham’s F12 medium with 2 mM L-

14

glutamine (500 ml bottles) with 10% Fetal Bovine Serum (Gemini Bio-Products,
Sacramento, CA) supplemented with 1% Penicillin (100 U/ml) and Streptomycin (100
µg/ml) (Invitrogen, Carlsbad, CA), 0.01 mg/ml Bovine pituitary extract, 0.005 mg/ml
insulin, 2.5 ng/ml of insulin like growth factor, 0.00125 mg/ml transferrin, 2.5 ng/ml
epidermal growth factor (Sigma Aldrich, St. Louis, MO) in 75 cm
2
flask (Nunc,
Portsmouth, NH). Cells were incubated at 37 °C in humidified air with 5% CO
2
.
When required for assays, confluent monolayers of RLE6TN cells were washed
twice with Dubelco’s Phophate buffered saline (PBS, Mediatech, Manassas, VA) and
then detached by treating with Trypsin EDTA (Gibco) for 3 min. Trypsin was then
neutralized by adding 7 ml of F12 media containing 10% FBS. The cells were
centrifuged at 125 g for 5 min and then re-suspended in fresh F12 with 10% FBS. Cells
were seeded into 6 well plates (Nunc, Portsmouth, NH) at a density of 5 x 10
6
cells per
well containing 2 ml of media and incubated overnight for treatment the next day.
Exposure to CSE (0.5 to 5%), acrolein (5 to 50µM), or nitric oxide (0.1 to 1 µM) was
carried out for 4- or 24 h time intervals at 37°C and 5% CO
2
.

Exposure of mice to cigarette smoke
Male A/J mice (Jackson Laboratories) 12 weeks of age were divided into two
groups, each composed of four

animals. The control group was kept in a filtered air
environment, whereas the remaining mice were exposed whole body to

the smoke
generated by Kentucky 3R4F reference cigarettes (Tobacco

Research Institute, University

15

of Kentucky, Lexington, KY),

having a declared content of 11 mg of total particulate
matter

and 0.73 mg of nicotine each. Before use, the cigarettes were

kept for 48 h in a
standardized atmosphere humidified with a

mixture of 70% glycerol and 30% water. The
smoke concentration and the duration of exposure were gradually increased in the first
week to get the mice acclimatized to the tobacco smoke.
A mixture of sidestream smoke (89%) and mainstream smoke (11%),

mimicking
an exposure to ETS, was produced by using a smoking

machine (model TE-10; Teague
Enterprises, Davis, CA), where

each smoldering cigarette was puffed for 2 s, once every
min

for a total of nine puffs. The machine was adjusted

to burn six cigarettes at one time,
6 h a day, 5 days a week for the total study period of 14 days. Sidestream

and mainstream
smokes were aspirated in a mixing chamber before

distribution to two exposure
chambers. Under those conditions,

the total particulate matter in the exposure chambers
was 80-90 mg/m
3
. The position

of the cages in the exposure chambers was rotated daily.
Isolation of lung mitochondria
Lung mitochondria were isolated using the method of differential centrifugation.
The mice were sacrificed and the lung excised. The lungs were rinsed in 2 ml of
mitochondrial isolation buffer (MIB) (Sucrose 250 mM, HEPES 20 mM, EDTA 1 mM,
EGTA 1 mM, Protease inhibitor 0.25%, BSA 0.5% pH 7.4) and then homogenized in 10
ml of MIB with a homogenizer. The homogenate was then centrifuged at 3110 rpm for 5
min at 4°C to remove tissue debris and nuclei, and yield a supernatant enriched in
mitochondria. The pellet was re suspended in 10 ml and the above process repeated until

16

no visible pellet was seen. The supernatant collected was then centrifuged at 12,500 rpm
for 10 min at 4°C to isolate mitochondria. To remove any cytosolic contaminants, the
mitochondrial pellet was re-suspended in MIB and again pellet by centrifugation at 9000
rpm for 8 min at 4°C
73
.
Cell viability assay
Cell viability assays were performed using MTT. In this assay cells were treated
with CSE, acrolein, or nitric oxide for 4 h to 24 h. After treatment, the cells were washed
with DPBS twice to remove stress and then replaced with 2 ml/well Hepes buffer
containing 0.5 mg/ml of MTT (Sigma Aldrich, St. Louis, MO). The cells were incubated
for 90 min at 37°C 5% CO
2
. After incubation the MTT solution was removed and 1 ml of
DMSO (Sigma Aldrich, St. Louis, MO) was added to dissolve the formazan crystals. The
plate was then read at 490 nm in a microplate spectrophotometer.

Western blots analyses
Western blot analysis was performed by washing the cells twice with DPBS and
then scraping the wells with a 1:1 mixture of non-reducing sample buffer (Pierce,
Rockford, IL) and RIPA (50 Mm Tris.HCl (pH 7.4)), lysis buffer containing protease
inhibitor, phosphatase inhibitor, DNAse and RNAse. 20 µg of sample was loaded per
well on 15% SDS PAGE gel. The protein was then transferred onto PVDF membrane
using the criterion system (Bio-Rad, Hercules, CA) and then probed against anti-
glutathione antibody (1:500) obtained from Virogen (Watertown, MA) for 48 h at 4°C,
and then against goat anti-mouse HRP conjugated antibody (Santa Cruz Biotechnology,

17

Santa Cruz, California) at 1:5000 dilution at RT for 90 min. The bands were visualized
using ECL kit from Thermoscientific (Rockford, IL).

Seahorse XF-24 metobolic flux analysis
RLE6TN cells were cultured on Seahorse XF-24 plates at a density of 40,000 K
cells per well the day before in situ measurements. On the day of in situ measurements
cells were treated with varying concentrations of CSE for 4 h and oxidative
phosphorylation and glycolysis were measured as according to Wu et
67
, using the
Seahorse XF24 Extracellular Flux analyzer (Seahorse Bioscience, North Billerica, MA,
USA). OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) were
normalized for protein concentration. The mitochondrial inhibitors used were oligomycin
(1µM), FCCP (1 µM), and rotenone (1 µM).

Enzyme activity assays
GAPDH activity - GAPDH activity in RLE-6TN cells was measured according to Ghezzi
et al
68
, with some modifications: cells were lysed in GAPDH reaction buffer (100 mM
Tris/HCl, 5 mM sodium arsenate, pH 8.6) 50 µl/well with the addition of protease
inhibitors and freezed-thawed 3 times post treatment. The protein concentration was
measured using Bradford assay. GAPDH activity was detected in the supernatant through
monitoring the increase in NADH formation spectrophotometrically at 340 nm in the
presence of 250 μM NAD
+
and 50 mg/ml glyceraldehyde-3-phosphate.

18

Glucose-6-Phosphate Dehydrogenase (G6PD) activity – G6PD activity in RLE6TN cells
was measured according to García-Nogales et al
69
with some modifications: cells were
lysed in reaction buffer (100 mM Tris/HCl, 5 mM sodium arsenate, pH 8.6) 50 µl/well
with the addition of protease inhibitors and freezed-thawed three times post treatment.
The protein concentration was measured using Bradford assay. 100 µg of protein was
mixed with the reaction mixture containing 100 µM NADP, 3.4 mM glucose 6-phosphate
and reaction buffer to a final volume of 1 ml. The absorbance was monitored at 340 nm
for 300 seconds. Enzyme activity was calculated from the slopes and expressed in
nmol/min/mg of protein.

Quantitation of pyridine nucleotides in RLE 6TN cells and lung mitochondria
HPLC analysis of NAD
+
, NADH, NADP
+
, NADPH was done according to
Klaidman et al
70
with modifications. An HPLC ZORBAX C
18
analytical column (5uM,
4.5×250mm) and a guard column (Agilent Technologies, Santa Clara, CA) were used for
analysis in an Agilent 1100 series HPLC system and a Hitachi Fluorescence
Spectrophotometer. All other chemicals were obtained from Sigma. An HPLC gradient
time program was used with 100% ammonium acetate (0.2M, pH 5.5) and 0% HPLC
grade methanol as initial condition followed by increasing concentration of methanol to
6.8 % over time. Pure NADH, NAD
+
,

NADPH, and NADP
+
at a concentration of 1
mg/ml were used for standardization of the method. The pyridine nucleotides were
quantified using the fluorescence spectrophotometer at excitation 330 nm and emission
460 nm. This was repeated several times to make a standard curve.

19

The cells after treatment were scraped in 50 µl of 0.2 M KCN/well prior to
chloroform extraction that was carried out at 14000 rpm in a microcentrifuge at 4
0
C. The
aqueous supernatant was collected, containing the soluble pyridine nucleotides and
extracted thrice to remove the lipids and proteins. The aqueous solution is then filtered
through a 0.45 µm positively charged filter (Pall Life Sciences, Port Washington, NY) to
remove the DNA and RNA. On diluting the filtrate with 0.2 M ammonium acetate it was
injected on a HPLC C
18
column.

Quantitation of thiols in RLE 6TN cells and lung mitochondria
GSH, GSNO, and GSSG were detected using HPLC with a coulometric
electrochemical detector from ESA (Chelmsford, MA). Electrochemical detection has
commonly been used to measure GSH and GSSG with HPLC (Han et al., 2006a; Harvey
et al., 1989; Rebrin et al., 2007) ESA offers CoulArray systems that utilize between 4 and
16 channels. A 4-channel electrochemical array was employed for the simultaneous
detection of GSH, GSNO, and GSSG. The mobile phase for isocratic elution of the
sulfhydryls was composed of 25 mM monobasic sodium phosphate, 0.5 mM 1-octane
sulfonic acid (ion-pairing agent), and 2.7% acetonitrile, pH 2.7. All chemicals including
GSH, GSSG, and GSNO were purchased from Sigma Chemicals (St. Louis, MO, USA).
The pH for the mobile phase should be adjusted with 85% phosphoric acid. A flow rate
of 1 ml/min was used with a C-18 column (5 µM , 4.6 × 250 mm). Acetonitrile is the key
component in modulating the retention times of GSH, GSNO, and GSSG. With 2.7% of
acetonitrile in the mobile phase, the retention times for GSH generally appears at ~6.5

20

min, GSNO ~19 min, and GSSG ~28.5 min (retention times also vary with the type of
column used). Increasing acetonitrile levels in the mobile phase will decrease the elution
time of the sulfhydryls, and conversely decreasing acetonitrile levels lengthen the
retention time of all sulfhydryls, particularly GSNO and GSSG. Initially standard
chemicals were used to create a calibration curve of respective thiols. Each pure chemical
was injected thrice and an average was used to create to calculate the respective thiols
concentration. Increasing potentials of 500 mV, 700 mV and 900 mV were applied on
three channels. GSH was monitored and detected on 500 mV and 700 mV; whereas
GSSG and GSNO were detected on 900 mV. The standards were repeatedly injected
between samples to ensure elution times.
The cells after treatment were scraped in 50 µl of 5% meta-phosphoric acid
containing 25mM ammonium sulfamate for precipitation of proteins. The precipitated
proteins were centrifuged at 14000 rpm for 10 min in a microcentrifuge. The supernatant
was collected and filtered further using pall 0.45 µm positively charged filter. The
samples were then stored at -70°C until analysis.

21

RESULTS
Effects of CSE, acrolein, and nitric oxide on cell viability in RLE6TN cells

Fig 5. Dose dependent effects of acrolein, nitric oxide, and CSE (short-term and long-
term) on cell viability measured by MTT assay in RLE6TN cells. (A) RLE6TN cell
viability with 24-h acrolein exposure. (B) RLE6TN cell viability with 24-h nitric oxide
(C) RLE6TN cell viability with 4 h CSE exposure. Data are the mean ± SD of data from
three separate experiments, each performed in duplicates. D) RLE6TN cell viability with
24 h CSE exposure. Data are the mean ± SD of data from three separate experiments,
each performed in duplicates. *P ≤ 0.05, ** P ≤ 0.01 versus control.

0
20
40
60
80
100
Control 10 20 30 40 80
% MTT
reduction
[Acrolein] (µM)
24 h treatment A

22

(Fig 5, Continued)

0
20
40
60
80
100
Control 0.1 0.5 1 2.5 5
% MTT
reduction
[NO]ss (µM)
24 h treatment
0
20
40
60
80
100
Control 0.5 1 2.5 5
% MTT
reduction
[CSE] (%)
**
**
4 h treatment
C
B
**

*

**

**

**

23

(Fig 5, Continued)

RLE6TN cells were exposed to 10, 20, 30, 40, 80 µM acrolein for 24 h. Figure
5A shows that acrolein causes a dose-dependent decrease in cell viability. Similarly,
nitric oxide at concentrations of 0.1, 0.5, 1.0, 2.5, and 5 µM shows a dose-dependent
decrease (Figure 5B). We tried to compare the effects of these oxidants with the effect of
CSE on cell viability at various concentrations. Short-term (4 h) CSE caused a significant
decrease in cell viability at 2.5 and 5% as did long-term (24 h) (Figure 5C and D).
0
20
40
60
80
100
Control 0.5 1 2.5 5
% MTT
reduction
[CSE] %
24 h treatment
D
** **
*
*

24

Effect of CSE on mitochondrial respiration and glycolysis in RLE6TN cells

Fig 6. CSE treatment leads to impairment of oxidative phosphorylation and a shift
towards glycolysis. A) OCR from RLE6TN cells treated with 0.5 and 1.75% CSE was
compared with OCR from untreated cells. Vertical lines indicate the time of addition of
mitochondrial inhibitors (A) oligomycin (1 µM), (B) FCCP (1 µM), or (C) rotenone (1
µM). The maximal respiratory capacity (following the addition of FCCP) is significantly
lower in 1.75% CSE treated cells as compared to untreated cells. B) The OCR/ECAR
ratio showed a shift towards glycolysis on 1.75% CSE treatment in RLE6TN cells.

A

25

(Fig 6: Continued)

CSE contains a large number of electrophiles and free radicals
11
, so we expected
to see some effect on mitochondrial respiration as the cell viability decreases. We did this
using a Seahorse XF-24 metabolic flux analyzer, which can quantify physiological
changes in mitochondrial respiration and glycolysis as a function of CSE. Oxygen
consumption rate (OCR), which is a direct indicator of mitochondrial oxidative
phosphorylation, was measured in the presence and absence of CSE. Because the method
is non-invasive, modulating the external environment of the cells with chemicals that
interfere with the normal mitochondrial respiration, can help monitor the effect of CSE
on the electron-transport chain.
B

26

Oligomycin is an inhibitor of ATP synthase that leads to inhibition of state 3
respiration. This is due to the blockage of the proton channel that causes the oxidative
phosphorylation of ADP to ATP. Carbonyl cyanide-p-trifluoromethoxy phenylhydrazone
commonly known as FCCP is an ionophore used to uncouple oxidation from
phosphorylation. It increases the permeability of cell membranes to protons, thus
relieving the mitochondria of ATP synthesis and allowing the mitochondria to respire at
its maximal capacity. Rotenone inhibits the transfer of electrons from the NADH
dehydrogenase to ubiquinone, thus inhibiting complex I activity, thus leading to a
complete shutdown of mitochondrial respiration.
As shown in Fig 5A the maximal respiratory capacity of mitochondrion decreases
significantly on addition of FCCP in cells treated with 1.75 % of CSE. This indicates a
decrease in the overall mitochondrial capacity to respire in presence of CSE. This may be
due to the effects of oxidants in cigarette smoke extract on cytochrome oxidase which is
responsible for the transfer of electrons from cytochrome c to molecular oxygen. As the
oxygen consumption rate is decreased complex IV is the most sensible target for cigarette
smoke extract.
Extracellular acidification rate (ECAR) is the rate of lactic acid production in the
cell. As shown in Fig 5B the OCR/ECAR ratio decreases on addition of the uncoupler
FCCP in 1.75% CSE treated cells as compared to control, thereby suggesting that cells is
increasingly dependent on anaerobic respiration, i.e., a shift towards glycolysis for the
generation of ATP.

27

Effect of CSE treatment on NADPH/NADP
+
and NADH/NAD
+
ratios

Fig 7. A) & B) Short-term (4h) CSE treatments lead to an increase in the
NADPH/NADP
+
, with no effect on NADH/NAD
+
ratio. C) & D) Prolonged (24h) CSE
treatment leads to a decrease in both NADPH/NADP
+
and NADH/NAD
+
ratio in
RLE6TN cells.

0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Control 0.5 1 2.5 5
NADPH/NADP
[CSE] (%)
4 h treatment
A

28

(Fig 7, Continued)

0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
Control 0.5 1 2.5 5
NADH/NAD
[CSE] (%)
4 h treatment
B
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Control 0.5 1 2.5
NADPH/NADP
[CSE] (%)
24 h
C
24 h
C

29

(Fig 7, Continued)

The redox status of the cell is partly based on the NADPH/NADP
+
and NADH/NAD
+

ratios. NADPH is the major reducing equivalent for a number of reactions in the cell in
order to counteract the damage caused by oxidative stress. Under normal conditions the
intracellular [NADP
+
] and [NAD
+
] are low, whereas their reduced equivalents are high
71

Short-term (4 h) CSE treatment leads to an increase in the NADPH/NADP
+
, which
may be part of a compensation mechanism to cope with the increasing oxidative stress,
while long term (24 h) exposure leads to a decrease in the ratio. There was no significant
effect on the NADH/NAD
+
ratio at 4 h but the ratio decreased at 24 h. This decrease may
be implicated to affect the biosynthesis, detoxification and other processes but need
further studies.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
Control 0.5 1 2.5
NADH/NAD
[CSE] (%)
24 h
D
24 h
D

30

Effect of CS treatment on NADPH/NADP
+
and NADH/NAD
+
ratios in male A/J mice
lung mitochondria

NADPH/NADP
+
NADH/NAD
+

Energized Control 0.82 0.6

2 weeks
smoke
exposed
1.41 1.33

Not
Energized Control 0.72 0.0243

2 weeks
smoke
exposed
0.71 0.0214

Table I. 2 weeks cigarette smoke exposed lung mitochondria shows an increase in
NADPH/NADP
+
and NADH/NAD
+
ratios when energized using glutamate/malate. When
not energized no significant decrease was found in the NADPH/NADP
+
with a slight
decrease in the NADH/NAD
+
.
The short-term exposure to cigarette smoke showed a similar trend in the
NADPH/NADP
+
and NADH/NAD
+
ratios in A/J mice as seen with the RLE-6TN cells.
This indicates the antioxidant capacity of the lungs that compensates for the increasing
oxidative stress. Glutamate/Malate was added to the mitochondria in order to energize
them. The presence of substrates gives us a better idea about the state of mitochondria as

31

it mimics the conditions in vivo and in the absence of G/M there is no change in the
NADPH/NADP
+
.

Effect of CSE treatment on GSH/GSSG ratios and GSNO levels

Fig 8. A) Short term (4h) CSE treatment leads to an increase in the GSH/GSSG ratio
whereas B) long term (24h) CSE treatment leads to a decrease in the GSH/GSSG ratio in
RLE-6TN cells. C) & D) GSNO levels after 4h and 24h CSE exposure respectively.

0
50
100
150
200
250
300
350
Control 0.5 1
GSH/GSSG
CSE [%]
4 h treatment
A

32

(Fig 8: Continued)

0
20
40
60
80
100
120
140
160
Control 0.5 1 1.75 2.5
GSH/GSSG
CSE[%]
24 h treatment
0
0.05
0.1
0.15
0.2
0.25
0.3
Control 0.5 1 1.75 2.5
GSNO nmol/mg
CSE [%]
4 h treatment
B
C

33

(Fig 8: Continued)

The GSH/GSSG is another redox couple that is used to define the redox environment of
the cell. The GSH/GSSG ratio is found to increase after 4 h CSE exposure, in agreement
with increases in the NADPH/NADP ratio. After 24 h CSE exposure the GSH/GSSG
ratio decreases in a dose-dependent manner. This indicates that the cells are able to
compensate the oxidative stress when exposed for a short period of time but with long-
term exposure the oxidative stress becomes too high for the cell to cope with. The GSNO
levels indicate an increase in nitrosylation after 24 h exposure as compared to 4 h
exposure. This indicates proteins may be susceptible to post translational modifications
after 24 h exposure to CSE.

0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Control 0.5 1 1.75 2.5
GSNO nmol/mg
CSE [%]
24 h treatment
D

34

Effect of CS treatment on GSH/GSSG ratio and GSNO in male A/J mice lung
mitochondria

GSH GSSG GSH/GSSG GSNO

Energized Control 0.539 0.21 2.49 0.063

2 weeks
smoke
exposed
0.55 0.12 4.51 0.073

Not
Energized
Control 0.075 0.62 0.12 -

2 weeks
smoke
exposed
0.12 0.5 0.23 -

Table II. 2 weeks cigarette smoke exposed lung mitochondria shows an increase in
GSH/GSSG ratio and GSNO levels when energized using glutamate/malate. The ratio
increased even in the absence of G/M.

The ratio of GSH/GSSG was found to increase on 2 week smoke exposure in the lung
mitochondria indicating that short term smoke exposure generates oxidative stress in the
mitochondria and there is increased synthesis of GSH in the mitochondria. Similar trend
was observed in the cytosol and the homogenate (data not shown). The increase in GSNO
indicates increase in the nitosylation of glutathione.

35

Glutathionylation on exposure to CSE
Glutathionylation is the major post-translational modification found upon
exposure to cigarette smoke extract. The most important physiological role of protein
glutathionylation is to provide an antioxidant defense to the proteins.

Fig 9. Western blot from 4h CSE exposed RLE 6TN cell extract.
Exposed redox-sensitive protein thiols can react with reactive oxygen species and
lead to formation of sulfinic acid or sulfonic acid which is irreversible modifications as
shown in Fig. 1. The rapid reaction of these protein thiyl radicals with GSH, however,
can prevent the formation of these higher oxidation states
24
. Two proteins of around 22
and 40 kD were found to be glutathionylated in a dose-dependent manner as shown by
the western blot.

[CSE]

36

Glucose 6 Phosphate dehydrogenase and Glyceraldehyde 3 Phosphate dehydrogenase
enzyme activity
Glucose 6 Phosphate dehydrogenase is one of the major enzymes of the pentose
phosphate pathway. It is also the most important source of NADPH in the cells. It can be
the one responsible for the increase in NADPH in RLE 6TN cells exposed to CSE. The
pentose phosphate pathway is known to be activated under oxidative stress. Thus
cigarette smoke may lead to the activation of the pentose phosphate pathway, leading to
increased activity of glucose 6 Phosphate dehydrogenase, which in effect leads to the rise
in NADPH. The same trend was also observed in 2 week smoke-exposed male A/J mice.

Fig 10. A). Glucose 6 Phosphate dehydrogenase enzyme activity from RLE-6TN cells
exposed to CSE for 4 h. B). Glucose 6 phosphate dehydrogenase enzyme activity from 2
weeks smoke exposed male A/J mice lung cytosol.
0
100
200
300
400
500
600
700
800
900
Control 0.5 1.75
G6PD activity nmol/min/mg
CSE [%]
RLE-6TN cells RLE-6TN cells
A

37

(Fig 10: Continued)

Glyceraldehyde 3 Phosphate dehydrogenase is an important enzyme of the
glycolytic pathway that catalyzes the conversion of glyceraldehyde-3-Phosphate to 1,3-
bisphospho-glycerate. Under oxidative stress, inactivation of glyceraldehyde-3P-
dehydrogenase results in re-routing of the metabolic flux from glycolysis to the pentose
phosphate pathway
74
.

0
100
200
300
400
500
600
700
800
900
1000
Control 2 weeks smoked
G6PD activity nmol/min/mg
2 week smoke exposed mice
B

38

Fig 11. A) Glyceraldehyde 3 Phosphate dehydrogenase enzyme activity form 4 h CSE
exposed RLE-6TN cell extract. B). Glyceraldehyde 3 Phosphate dehydrogenase enzyme
activity form 2 week smoke exposed male A/J mice lung cytosol.

0
50
100
150
200
250
300
Control 0.5 1.75
GAPDH activity nmol/s/mg
[CSE] (%)
RLE-6TN cells
0
2000
4000
6000
Control 2 week Smoked
GAPDH Activity nmol/min/mg
2 week smoke exposed mice
B

39

It is evident from the in vitro data that GAPDH is slightly inactivated upon exposure to
CSE. But this is not the case in A/J mice. This might be because of the short duration of
smoke exposure, which may not be sufficient to induce a major change in the metabolic
function of the cells.

40

DISCUSSION
Chronic obstructive pulmonary disease is still not completely treatable, though the
general understanding of the pathophysiology has improved. The above study helps
understand the timeline of the development of COPD. Understanding the dynamics of the
changes in the redox status of the cell upon exposure to cigarette smoke has been one of
the preliminary aims of the study. This would help assess the behavioral kinetics and
risks associated with cigarette smoke exposure.
RLE-6TN cells exposed to increasing concentrations of CSE for 4 h and 24 h
showed a dose dependent decrease in cell viability (Fig 5C & D). Acrolein and nitric
oxide (components of CSE) also showed a similar dose dependent decrease in cell
viability after 24h exposure( Fig 5A & B). Oxygen consumption rate was also sensitive to
CSE in a dose dependent manner and preceded cell death which correlates with the cell
viability data (Fig 6A). The OCR/ECAR ratio signifies that the cells are becoming less
dependent on the mitochondria for ATP and more on the glycolytic pathways in the
cytosol (Fig 6B).
The cell’s redox status is essential to maintain proteins in a reduced state and to
function at maximal capacity. The perturbation in this redox status leads to protein
dysfuntion and a perturbed redox signaling. Acrolein, nitric oxide, peroxynitrile, and
various other components can be expected to modulate the redox status of the cells. The
main response we saw after 4 h CSE exposure was an increase in NADPH/NADP ratio.
At the same time, 24 h exposure caused a decrease in NADPH/NADP ratio (Fig 7A & C)
in RLE-6TN cells. This indicates the ability of the cells to compensate for the oxidative

41

stress by rerouting the metabolic flux through the pentose phosphate pathway, thus
generating more reducing pyridine nucleotides in the form of NADPH on short-term
exposure. This was also the case with A/J mice exposed to 2 weeks cigarette smoke
(Table I). The findings were also corroborated by the GSH/GSSG ratios that showed an
increase in the ratio after 4 h exposure (Fig 8A). This was also seen in the in vivo studies
(Table II). Male A/J mice have shown to develop COPD pathology after 6-16 weeks of
smoke exposure. Thus 2 week smoke exposure is early in the study. Nevertheless, the
timeline of COPD development can be ascertained from a redox perspective using these
findings.
Protein post translational modification can lead to changes in activity,
conformation, stability and hence the function
67
. Since cigarette smoke is known to
contain reactive oxygen and nitrogen species, it is likely that CS exposure to culture cells
and in vivo will lead to protein post translational modifications. The extent of effect on
protein function will depend on the amino acids which are altered and the extent of
modification. The levels of nitrosylated glutathione (GSNO) are seen to increase in RLE-
6TN cells and also in vivo when exposed to cigarette smoke (Fig 8 C & D, Table II). In
case of 24h CSE exposed RLE-6TN cells the increase is seen with higher concentration
than with 4h CSE exposure. This indicates that this protein modification may be
reversible and the cells may be able to recover from the modification.
Glutathionylation of proteins is another post translational modification which
helps to protect the proteins against the deleterious effects of increased oxidative stress.
Two proteins were found be increasingly glutathionylated with increasing concentration

42

of CSE exposure in RLE-6TN cells (Fig. 9). Thus the alteration in the redox status of the
cells leads to extensive protein glutathionylation which can be described as a protective
mechanism developed by the cells. The modification being reversible, the protein can
function in its normal capacity in the absence of stress.
Glucose-6-Phosphate Dehydrogenase is the first enzyme in the pentose phosphate
pathway which is known to be activated by nitrosative stress
53
. The increase in the
activity of G6PD (Fig 10 A & B) after cigarette smoke exposure may be responsible for
the increase in NADPH/NADP ratio. The inactivation of GAPDH after 4h CSE exposure
(Fig 11 A) indicates changes in the cytoplasmic metabolites for balancing the redox
status of the cells
74
. Other enzymes like isocitrate dehydrogenase, malic enzyme and
aldehyde dehydrogenase may also be responsible for the generation of NADPH. But this
needs to be probed further.
Nicotinamide nucleotide transhydrogenase is another enzyme which may be
responsible for the increase in NADPH. Its role in the mitochondria is still under study
and will need further studies. If responsible it may also account for the decrease in the
NADH which we see after CSE exposure in RLE-6TN cells since the major role of this
enzyme is as a proton translocating transhydrogenase involved in the conversion of
NADP to NADPH.

43

REFERENCES
1. Mathers CD, Loncar D, 2006 Projections of Global Mortality and Burden of
Disease from 2002 to 2030. PLoS
Med 3(11): e442.doi:10.1371/journal.pmed.0030442
2. National Health Interview Survey, 2007, Hyattsville, MD: National Center for
Health Statistics. Unpublished data tabulated by NHLBI, 2009.
3. Mannino DM, Homa DM, Akinbami LJ, et al. Chronic Obstructive Pulmonary
Disease Surveillance—United States, 1971–2000. MMWR. 2002;51(SS06):1-16.
Available at: http://www.cdc.gov/mmwr/preview/mmwrhtml/ss5106a1.htm.

4. Pauwels RA, Buist AS, Calverley PMA, et al. 2001. Global strategy for the
diagnosis, management, and prevention of chronic obstructive pulmonary disease.
NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD)
workshop summary. Am J Respir Crit Care Med, 163:1256.

5. Kumar P, Clark M (2005). Clinical Medicine, 6ed. Elsevier Saunders. pp 900-
901.

6. Yoshida T, Tuder R M, 2007, Pathobiology of cigarette smoke-induced chronic
obstructive pulmonary disease. Physiol Rev 87: 1047–1082.

7. Rangasamy, T., Misra, V., Zhen, L., Thankersely, C. G., Tuder, R. M., and
Biswal, S. (2009). Cigarette smoke-induced emphysema in A/J mice is associated
with pulmonary oxidative stress, apoptosis of lung cells, and global alterations in
gene expression. Am. J. Physiol. Lung Cell. Mol.Physiol. 296, L888–L900.

8. Repine, J.E., Bast, A. & Lankhorst, I. Oxidative stress in chronic obstructive
pulmonary disease. Oxidative Stress Study Group. Am J Respir Crit Care Med
156, 341-357 (1997).

9. Church, D.F. & Pryor, W.A. Free-radical chemistry of cigarette smoke and its
toxicological implications. Environmental health perspectives 64, 111-126
(1985).

10. Ou, B. & Huang, D. Fluorescent approach to quantitation of reactive oxygen
species in mainstream cigarette smoke. Analytical chemistry 78, 3097-3103
(2006).

11. Halliwell, B. & Gutteridge, J.M.C. The chemistry of free radicals and related
'reactive species'. in Free Radicals in Biology and Medicine (Oxford University
Press, Inc, United States, 2004).

44

12. Stevenson, C.S., Koch, L.G. & Britton, S.L. Aerobic capacity, oxidant stress, and
chronic obstructive pulmonary disease--a new take on an old hypothesis.
Pharmacology & therapeutics 110, 71-82 (2006).

13. Griffith, O.W. Biologic and pharmacologic regulation of mammalian glutathione
synthesis. Free radical biology & medicine 27, 922-935 (1999).

14. Han, D., Canali, R., Rettori, D. & Kaplowitz, N. Effect of glutathione depletion
on sites and topology of superoxide and hydrogen peroxide production in
mitochondria. Molecular pharmacology 64, 1136-1144 (2003).

15. Schafer, F.Q. & Buettner, G.R. Redox environment of the cell as viewed through
the redox state of the glutathione disulfide/glutathione couple. Free radical
biology & medicine 30, 1191-1212 (2001).

16. Han, D., Hanawa, N., Saberi, B. & Kaplowitz, N. Mechanisms of liver injury. III.
Role of glutathione redox status in liver injury. American journal of physiology
291, G1-7 (2006).

17. Antunes, F. & Cadenas, E. Cellular titration of apoptosis with steady state
concentrations of H(2)O(2): submicromolar levels of H(2)O(2) induce apoptosis
through Fenton chemistry independent of the cellular thiol state. Free radical
biology & medicine 30, 1008-1018 (2001).

18. Baglole, C.J., et al. Differential induction of apoptosis by cigarette smoke extract
in primary human lung fibroblast strains: implications for emphysema. Am J
Physiol Lung Cell Mol Physiol 291, L19-29 (2006).

19. Rahman, I. & Adcock, I.M. Oxidative stress and redox regulation of lung
inflammation in COPD. Eur Respir J 28, 219-242 (2006).

20. Serviddio, G., et al. Evidence of lower oxidative stress in the air spaces of patients
with reversible COPD. International journal of immunopathology and
pharmacology 19, 617-628 (2006).

21. Martinez-Ruiz, A. & Lamas, S. S-nitrosylation: a potential new paradigm in
signal transduction. Cardiovascular Research 62, 43-52 (2004).

22. Cumming, R.C., et al. Protein disulfide bond formation in the cytoplasm during
oxidative stress. J Biol Chem 279, 21749-21758 (2004).

45

23. Klatt, P. & Lamas, S. Regulation of protein function by S-glutathiolation in
response to oxidative and nitrosative stress. Eur J Biochem 267, 4928-4944
(2000).

24. Hurd TR, Costa NJ, Dahm CC, Beer SM, Brown SE, Filipovska A, Murphy MP.
Glutathionylation of mitochondrial proteins. Antioxid Redox Signal. 2005 Jul-
Aug;7(7-8):999-1010.

25. Boveris A., Oshino, N. & Chance, B. The cellular production of hydrogen
peroxide. Biochem J 128, 617-630 (1972).

26. Boveris, A. & Cadenas, E. Mitochondrial production of superoxide anions and its
relationship to the antimycin insensitive respiration. FEBS Lett 54, 311-314
(1975).

27. Cadenas, E., Boveris, A., Ragan, C.I. & Stoppani, A.O. Production of superoxide
radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-
cytochrome c reductase from beef-heart mitochondria. Arch Biochem
Biophys 180, 248-257 (1977).

28. Boveris, A., Cadenas, E. & Stoppani, A.O. Role of ubiquinone in the
mitochondrial generation of hydrogen peroxide. Biochem J 156, 435-444 (1976).

29. Turrens, J.F. & Boveris, A. Generation of superoxide anion by the NADH
dehydrogenase of bovine heart mitochondria. Biochem J 191, 421-427 (1980).

30. Nichollas, D.G. & Ferguson, S.J. Superoxide production by complexes I and III.
in Bioenergetics 3 (Elsevier Science, London, 2002).

31. Han, D., Williams, E. & Cadenas, E. Mitochondrial respiratory chain-dependent
generation of superoxide anion and its release into the intermembrane space.
Biochem J 353, 411-416 (2001).

32. Antunes, F., Boveris, A. & Cadenas, E. On the mechanism and biology of
cytochrome oxidase inhibition by nitric oxide. Proceedings of the National
Academy of Sciences of the United States of America 101, 16774-16779 (2004).

33. Brown G.C, C.C.E. Nanomolar concentrations of nitric oxide reversibly inhibit
synaptosomal respiration by competing with oxygen at cytochrome oxidase.
FEBS Lett 356, 295-298 (1994).

46

34. Boveris, A., Costa, L.E., Cadenas, E. & Poderoso, J.J. Regulation of
mitochondrial respiration by adenosine diphosphate, oxygen, and nitric oxide.
Methods Enzymol 301, 188-198 (1999).

35. Moncada, S. & Erusalimsky, J.D. Does nitric oxide modulate mitochondrial
energy generation and apoptosis? Nat Rev Mol Cell Biol 3, 214-220 (2002).

36. Poderoso, J.J., et al. Nitric oxide inhibits electron transfer and increases
superoxide radical production in rat heart mitochondria and submitochondrial
particles. Archives of biochemistry and biophysics 328, 85-92 (1996).

37. Cardellach, F., Alonso, J.R., Lopez, S., Casademont, J. & Miro, O. Effect of
smoking cessation on mitochondrial respiratory chain function. Journal of
toxicology 41, 223-228 (2003).

38. Comhair, S.A., et al. Superoxide dismutase inactivation in pathophysiology of
asthmatic airway remodeling and reactivity. Am J Pathol 166, 663-674 (2005).

39. Brown, G.C. & Borutaite, V. Inhibition of mitochondrial respiratory complex I by
nitric oxide, peroxynitrite and S-nitrosothiols. Biochim Biophys Acta 1658, 44-49
(2004).

40. Han, D., et al. Sites and mechanisms of aconitase inactivation by peroxynitrite:
modulation by citrate and glutathione. Biochemistry 44, 11986-11996 (2005).

41. Murray, J., Taylor, S.W., Zhang, B., Ghosh, S.S. & Capaldi, R.A. Oxidative
damage to mitochondrial complex I due to peroxynitrite: identification of reactive
tyrosines by mass spectrometry. The Journal of biological chemistry 278, 37223-
37230 (2003).

42. Taylor, E.R., et al. Reversible glutathionylation of complex I increases
mitochondrial superoxide formation. The Journal of biological chemistry 278,
19603-19610 (2003).

43. Halestrap, A.P., Doran, E., Gillespie, J.P. & O'Toole, A. Mitochondria and cell
death. Biochem Soc Trans 28, 170-177 (2000).

44. Cadenas, E. Mitochondrial free radical production and cell signaling. Mol
Aspects Med 25, 17-26 (2004).

45. Demedts, I.K., Demoor, T., Bracke, K.R., Joos, G.F. & Brusselle, G.G.
Role of apoptosis in the pathogenesis of COPD and pulmonary emphysema.
Respiratory research 7, 53 (2006).

47

46. Desagher, S. & Martinou, J.C. Mitochondria as the central control point of
apoptosis. Trends Cell Biol 10, 369-377 (2000).

47. Zhong, C.Y., Zhou, Y.M., Joad, J.P. & Pinkerton, K.E. Environmental
tobacco smoke suppresses nuclear factor-kappaB signaling to increase
apoptosis in infant monkey lungs. American journal of respiratory and critical
care medicine 174, 428-436 (2006).

48. Gogvadze, V. & Orrenius, S. Mitochondrial regulation of apoptotic cell death.
Chemico-biological interactions 163, 4-14 (2006).

49. Toorn van der , M., et al. Cigarette smoke induced blockade of the
mitochondrial respiratory chain switches lung epithelial cell apoptosis into
necrosis. American journal of physiology (2007).

50. Slebos, D.J., et al. Mitochondrial Localization and Function of Heme Oxygenase-
1 in Cigarette Smoke-Induced Cell Death. Am J Respir Cell Mol Biol (2006).

51. Henson, P.M., Vandivier, R.W. & Douglas, I.S. Cell death, remodeling, and
repair in chronic obstructive pulmonary disease? Proceedings of the American
Thoracic Society 3, 713-717 (2006).

52. Wamelink, M. M., Struys, E. A., and Jakobs, C. (2008) The biochemistry,
metabolism and inherited defects of the pentose phosphate pathway: a review. J.
Inherit. Metab. Dis. 31, 703–717.

53. Bolaños JP, Delgado-Esteban M, Herrero-Mendez A, Fernandez-Fernandez S,
Almeida A, Regulation of glycolysis and pentose-phosphate pathway by nitric
oxide: impact on neuronal survival. Biochim Biophys Acta. 2008 Jul-
Aug;1777(7-8):789-93. Epub 2008 Apr 13. Review.

54. Bolaños Juan P. and Angeles Almeida, The Pentose-Phosphate Pathway in
neuronal survival against nitrosative stress, IUBMB Life, 62(1): 14–18, January
2010

55. Garcia-Nogales, P., Almeida, A., and Bolanos, J. P. (2003) Peroxynitrite protects
neurons against nitric oxide-mediated apoptosis. A key role for glucose-6-
phosphate dehydrogenase activity in neuroprotection. J. Biol.Chem. 278, 864–
874.

56. Lehninger A.L., D.L. Nelson, M.M. Cox, Principles of Biochemistry, 4
th
edition,
Worth Publishers Inc., New York, pg 549.

57. 3R4F research grade cigarette’s preliminary analysis available at
http://www.ca.uky.edu/refcig/3R4F%20Preliminary%20Analysis.pdf

48

58. Carp H, Janoff A. Possible mechanisms of emphysema in smokers. In vitro
suppression of serum elastase-inhibitory capacity by fresh cigarette smoke and its
prevention by antioxidants. Am Rev Respir Dis 118:617–621, 1978.

59. Yang Se-Ran, Jessica Wright, Mark Bauter, Kathryn Seweryniak, Aruna Kode
and Irfan Rahman, Sirtuin regulates cigarette smoke-induced proinflammatory
mediator release via RelA/p65 NF-B in macrophages in vitro and in rat lungs in
vivo: implications for chronic inflammation and aging, Am J Physiol Lung Cell
Mol Physiol, 292:567-576, 2007.

60. Li XY, Rahman I, Donaldson K, MacNee W.. Mechanisms of cigarette smoke
induced increased airspace permeability. Thorax.1996;51:465–471.

61. Rahman Irfan, Agnes Bel, Brigitte Mulier, Kenneth Donaldson and William
MacNee, Differential regulation of glutathione by oxidant and dexamethasone in
alveolar epithelial cells, Am J Physiol Lung Cell Mol Physiol 275:80-86, 1998.

62. Lannan S, Donaldson K, Brown D, MacNee W., Effect of cigarette smoke and its
condensates on alveolar epithelial cell injury in vitro. Am J Physiol. 1994
Jan;266(1 Pt 1):L92-100.

63. Arunachalam G, Yao H, Sundar IK, Caito S, Rahman I, SIRT1 regulates oxidant-
and cigarette smoke-induced eNOS acetylation in endothelial cells: Role of
resveratrol. Biochem Biophys Res Commun. 2010 Feb 26;393(1):66-72. Epub
2010 Jan 25.

64. Rajendrasozhan S, Hwang JW, Yao H, Kishore N, Rahman I, Anti-inflammatory
effect of a selective IkappaB kinase-beta inhibitor in rat lung in response to LPS
and cigarette smoke. Pulm Pharmacol Ther. 2010 Jan 18. [Epub ahead of print]

65. Lambert C, McCue J, Portas M, Ouyang Y, Li J, Rosano TG, Lazis A, Freed BM,
Acrolein in cigarette smoke inhibits T-cell responses. J Allergy Clin
Immunol. 2005 Oct;116(4):916-22. Epub 2005 Aug 1

66. Yang Se-Ran, Asiya S. Chida, Mark R. Bauter, Nusrat Shafiq, Kathryn
Seweryniak, Sanjay B. Maggirwar, Iain Kilty and Irfan Rahman, Cigarette smoke
induces proinflammatory cytokine release by activation of NF-B and
posttranslational modifications of histone deacetylase in macrophages, Am J
Physiol Lung Cell Mol Physiol 291: L46–L57, 2006

49

67. Wu M., Neilson A., Swift A.L., Moran RTamagnine J.,. Parslow D, Armistead
S.,. Lemire K, Orrell J., Teich J., Chomicz S.and Ferrick D.A., Multiparameter
metabolic analysis reveals a close link between attenuated mitochondrial
bioenergetic function and enhanced glycolysis dependency in human tumour
cells, Am. J. Physiol. Cell Physiol. 292 (2007), pp. C125–C136.

68. Ghezzi P, Romines B, Fratelli M, Eberini I, Gianazza E, Casagrande S, Laragione
T, Mengozzi M, Herzenberg LA, Herzenberg LA, Protein glutathionylation:
coupling and uncoupling of glutathione to protein thiol groups in lymphocytes
under oxidative stress and HIV infection, Mol Immunol. 2002 Feb;38(10):773-80.

69. García-Nogales P, Almeida A, Fernández E, Medina JM, Bolaños JP, Induction of
glucose-6-phosphate dehydrogenase by lipopolysaccharide contributes to
preventing nitric oxide-mediated glutathione depletion in cultured rat astrocytes, J
Neurochem. 1999 Apr;72(4):1750-8.

70. Klaidman, L.K., A.C. Leung, and J.D. Adams, High-Performance Liquid
Chromatography Analysis of Oxidized and Reduced Pyridine Dinucleotides in
Specific Brain Regions. Analytical Biochemistry, 1995. 228(2): p. 312-317.

71. Pollak N, Dolle C and Ziegler M, The power to reduce: pyridine nucleotides –
small molecules with a multitude of functions, Biochem. J. (2007) 402, 205–218

72. Velsor L, Kariya C, Kachadourian R, and Day B, Mitochondrial Oxidative Stress
in the Lungs of Cystic Fibrosis Transmembrane Conductance Regulator Protein
Mutant Mice, American Journal of Respiratory Cell and Molecular Biology. Vol.
35, pp. 579-586, 2006

73. Ralser M et al. (2007). "Dynamic rerouting of the carbohydrate flux is key to
counteracting oxidative stress.". J Biol 6 (11): 10

74. Francisco J. Corpas, Luis A. del Río and Juan B. Barroso, Post ‑translational
modifications mediated by reactive nitrogen species, Plant Signaling & Behavior
3:5, 301-303; May 2008

50

ALPHABETIZED BIBLIOGRAPHY

Antunes, F. & Cadenas, E. Cellular titration of apoptosis with steady state concentrations of
H(2)O(2): submicromolar levels of H(2)O(2) induce apoptosis through Fenton
chemistry independent of the cellular thiol state. Free radical biology & medicine 30,
1008-1018 (2001).
Antunes, F., Boveris, A. & Cadenas, E. On the mechanism and biology of cytochrome
oxidase inhibition by nitric oxide. Proceedings of the National Academy of Sciences
of the United States of America 101, 16774-16779 (2004).

Arunachalam G, Yao H, Sundar IK, Caito S, Rahman I, SIRT1 regulates oxidant- and
cigarette smoke-induced eNOS acetylation in endothelial cells: Role of resveratrol.
Biochem Biophys Res Commun. 2010 Feb 26;393(1):66-72. Epub 2010 Jan 25.

Baglole, C.J., et al. Differential induction of apoptosis by cigarette smoke extract in primary
human lung fibroblast strains: implications for emphysema. Am J Physiol Lung Cell
Mol Physiol 291, L19-29 (2006).

Bolaños Juan P. and Angeles Almeida, The Pentose-Phosphate Pathway in neuronal survival
against nitrosative stress, IUBMB Life, 62(1): 14–18, January 2010

Bolaños JP, Delgado-Esteban M, Herrero-Mendez A, Fernandez-Fernandez S, Almeida A,
Regulation of glycolysis and pentose-phosphate pathway by nitric oxide: impact on
neuronal survival. Biochim Biophys Acta. 2008 Jul-Aug;1777(7-8):789-93. Epub
2008 Apr 13. Review.

Boveris, A., Oshino, N. & Chance, B. The cellular production of hydrogen peroxide.
Biochem J 128, 617-630 (1972).

Boveris, A. & Cadenas, E. Mitochondrial production of superoxide anions and its
relationship to the antimycin insensitive respiration. FEBS Lett 54, 311-314 (1975).

Boveris, A., Cadenas, E. & Stoppani, A.O. Role of ubiquinone in the mitochondrial
generation of hydrogen peroxide. Biochem J 156, 435-444 (1976).

Boveris, A., Costa, L.E., Cadenas, E. & Poderoso, J.J. Regulation of mitochondrial
respiration by adenosine diphosphate, oxygen, and nitric oxide. Methods Enzymol
301, 188-198 (1999).

Brown G.C, C.C.E. Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal
respiration by competing with oxygen at cytochrome oxidase. FEBS Lett 356, 295-
298 (1994).

51

Brown, G.C. & Borutaite, V. Inhibition of mitochondrial respiratory complex I by nitric
oxide, peroxynitrite and S-nitrosothiols. Biochim Biophys Acta 1658, 44-49 (2004).

Cadenas, E., Boveris, A., Ragan, C.I. & Stoppani, A.O. Production of superoxide radicals
and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome
c reductase from beef-heart mitochondria. Arch Biochem Biophys 180, 248-257
(1977).

Cadenas, E. Mitochondrial free radical production and cell signaling. Mol Aspects Med 25,
17-26 (2004).

Cardellach, F., Alonso, J.R., Lopez, S., Casademont, J. & Miro, O. Effect of smoking
cessation on mitochondrial respiratory chain function. Journal of toxicology 41, 223-
228 (2003).

Carp H, Janoff A. Possible mechanisms of emphysema in smokers. In vitro suppression of
serum elastase-inhibitory capacity by fresh cigarette smoke and its prevention by
antioxidants. Am Rev Respir Dis 118:617–621, 1978.

Church, D.F. & Pryor, W.A. Free-radical chemistry of cigarette smoke and its toxicological
implications. Environmental health perspectives 64, 111-126 (1985).

Comhair, S.A., et al. Superoxide dismutase inactivation in pathophysiology of asthmatic
airway remodeling and reactivity. Am J Pathol 166, 663-674 (2005).

Cumming, R.C., et al. Protein disulfide bond formation in the cytoplasm during oxidative
stress. J Biol Chem 279, 21749-21758 (2004).

Demedts, I.K., Demoor, T., Bracke, K.R., Joos, G.F. & Brusselle, G.G. Role of
apoptosis in the pathogenesis of COPD and pulmonary emphysema. Respiratory
research 7, 53 (2006).

Desagher, S. & Martinou, J.C. Mitochondria as the central control point of apoptosis. Trends
Cell Biol 10, 369-377 (2000).

Francisco J. Corpas, Luis A. del Río and Juan B. Barroso, Post ‑translational modifications
mediated by reactive nitrogen species, Plant Signaling & Behavior 3:5, 301-303; May
2008

García-Nogales P, Almeida A, Fernández E, Medina JM, Bolaños JP, Induction of glucose-6-
phosphate dehydrogenase by lipopolysaccharide contributes to preventing nitric
oxide-mediated glutathione depletion in cultured rat astrocytes, J Neurochem. 1999
Apr;72(4):1750-8.

52

Garcia-Nogales, P., Almeida, A., and Bolanos, J. P. (2003) Peroxynitrite protects neurons
against nitric oxide-mediated apoptosis. A key role for glucose Gogvadze, V. &
Orrenius, S. Mitochondrial regulation of apoptotic cell death. Chemico-biological
interactions 163, 4-14 (2006).

Ghezzi P, Romines B, Fratelli M, Eberini I, Gianazza E, Casagrande S, Laragione
T, Mengozzi M, Herzenberg LA, Herzenberg LA, Protein glutathionylation: coupling
and uncoupling of glutathione to protein thiol groups in lymphocytes under oxidative
stress and HIV infection, Mol Immunol. 2002 Feb;38(10):773-80.

Griffith, O.W. Biologic and pharmacologic regulation of mammalian glutathione synthesis.
Free radical biology & medicine 27, 922-935 (1999).

Gogvadze, V. & Orrenius, S. Mitochondrial regulation of apoptotic cell death. Chemico-
biological interactions 163, 4-14 (2006).

Halestrap, A.P., Doran, E., Gillespie, J.P. & O'Toole, A. Mitochondria and cell death.
Biochem Soc Trans 28, 170-177 (2000).

Halliwell, B. & Gutteridge, J.M.C. The chemistry of free radicals and related 'reactive
species'. in Free Radicals in Biology and Medicine (Oxford University Press, Inc,
United States, 2004).

Han, D., Canali, R., Rettori, D. & Kaplowitz, N. Effect of glutathione depletion on sites and
topology of superoxide and hydrogen peroxide production in mitochondria.
Molecular pharmacology 64, 1136-1144 (2003).

Han, D., et al. Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation
by citrate and glutathione. Biochemistry 44, 11986-11996 (2005).

Han, D., Hanawa, N., Saberi, B. & Kaplowitz, N. Mechanisms of liver injury. III. Role of
glutathione redox status in liver injury. American journal of physiology 291, G1-7
(2006).

Han, D., Williams, E. & Cadenas, E. Mitochondrial respiratory chain-dependent generation
of superoxide anion and its release into the intermembrane space. Biochem J 353,
411-416 (2001).

Henson, P.M., Vandivier, R.W. & Douglas, I.S. Cell death, remodeling, and repair in chronic
obstructive pulmonary disease? Proceedings of the American Thoracic Society 3,
713-717 (2006).

53

Hurd TR, Costa NJ, Dahm CC, Beer SM, Brown SE, Filipovska A, Murphy MP.
Glutathionylation of mitochondrial proteins. Antioxid Redox Signal. 2005 Jul-
Aug;7(7-8):999-1010.

Klaidman, L.K., A.C. Leung, and J.D. Adams, High-Performance Liquid Chromatography
Analysis of Oxidized and Reduced Pyridine Dinucleotides in Specific Brain Regions.
Analytical Biochemistry, 1995. 228(2): p. 312-317.

Klatt, P. & Lamas, S. Regulation of protein function by S-glutathiolation in response to
oxidative and nitrosative stress. Eur J Biochem 267, 4928-4944 (2000).

Kumar P, Clark M (2005). Clinical Medicine, 6ed. Elsevier Saunders. pp 900-901.

Lambert C, McCue J, Portas M, Ouyang Y, Li J, Rosano TG, Lazis A, Freed BM, Acrolein
in cigarette smoke inhibits T-cell responses. J Allergy Clin Immunol. 2005
Oct;116(4):916-22. Epub 2005 Aug 1

Lannan S, Donaldson K, Brown D, MacNee W., Effect of cigarette smoke and its
condensates on alveolar epithelial cell injury in vitro. Am J Physiol. 1994 Jan;266(1
Pt 1):L92-100.

Lehninger A.L., D.L. Nelson, M.M. Cox, Principles of Biochemistry, 4
th
edition, Worth
Publishers Inc., New York, pg 549.

Li XY, Rahman I, Donaldson K, MacNee W.. Mechanisms of cigarette smoke induced
increased airspace permeability. Thorax.1996;51:465–471.

Martinez-Ruiz, A. & Lamas, S. S-nitrosylation: a potential new paradigm in signal
transduction. Cardiovascular Research 62, 43-52 (2004).

Mathers CD, Loncar D, 2006 Projections of Global Mortality and Burden of Disease from
2002 to 2030. PLoS Med 3(11): e442.doi:10.1371/journal.pmed.0030442
Mannino DM, Homa DM, Akinbami LJ, et al. Chronic Obstructive Pulmonary Disease
Surveillance—United States, 1971–2000. MMWR. 2002;51(SS06):1-16.
Moncada, S. & Erusalimsky, J.D. Does nitric oxide modulate mitochondrial energy
generation and apoptosis? Nat Rev Mol Cell Biol 3, 214-220 (2002).

Murray, J., Taylor, S.W., Zhang, B., Ghosh, S.S. & Capaldi, R.A. Oxidative damage to
mitochondrial complex I due to peroxynitrite: identification of reactive tyrosines by
mass spectrometry. The Journal of biological chemistry 278, 37223-37230 (2003).

54

National Health Interview Survey, 2007, Hyattsville, MD: National Center for Health
Statistics. Unpublished data tabulated by NHLBI, 2009.
Nichollas, D.G. & Ferguson, S.J. Superoxide production by complexes I and III. in
Bioenergetics 3 (Elsevier Science, London, 2002).

Ou, B. & Huang, D. Fluorescent approach to quantitation of reactive oxygen species in
mainstream cigarette smoke. Analytical chemistry 78, 3097-3103 (2006).

Pauwels RA, Buist AS, Calverley PMA, et al. 2001. Global strategy for the diagnosis,
management, and prevention of chronic obstructive pulmonary disease.
NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD)
workshop summary. Am J Respir Crit Care Med, 163:1256.

Poderoso, J.J., et al. Nitric oxide inhibits electron transfer and increases superoxide radical
production in rat heart mitochondria and submitochondrial particles. Archives of
biochemistry and biophysics 328, 85-92 (1996).

Pollak N, Dolle C and Ziegler M, The power to reduce: pyridine nucleotides – small
molecules with a multitude of functions, Biochem. J. (2007) 402, 205–218

Rahman, I. & Adcock, I.M. Oxidative stress and redox regulation of lung inflammation in
COPD. Eur Respir J 28, 219-242 (2006).

Rahman Irfan, Agnes Bel, Brigitte Mulier, Kenneth Donaldson and William MacNee,
Differential regulation of glutathione by oxidant and dexamethasone in alveolar
epithelial cells, Am J Physiol Lung Cell Mol Physiol 275:80-86, 1998.

Rajendrasozhan S, Hwang JW, Yao H, Kishore N, Rahman I, Anti-inflammatory effect of a
selective IkappaB kinase-beta inhibitor in rat lung in response to LPS and cigarette
smoke. Pulm Pharmacol Ther. 2010 Jan 18. [Epub ahead of print]

Rangasamy, T., Misra, V., Zhen, L., Thankersely, C. G., Tuder, R. M., and Biswal, S. (2009).
Cigarette smoke-induced emphysema in A/J mice is associated with pulmonary
oxidative stress, apoptosis of lung cells, and global alterations in gene expression.
Am. J. Physiol. Lung Cell. Mol.Physiol. 296, L888–L900.

Ralser M et al. (2007). "Dynamic rerouting of the carbohydrate flux is key to counteracting
oxidative stress.". J Biol 6 (11): 10

Repine, J.E., Bast, A. & Lankhorst, I. Oxidative stress in chronic obstructive pulmonary
disease. Oxidative Stress Study Group. Am J Respir Crit Care Med 156, 341-357
(1997).

55

Schafer, F.Q. & Buettner, G.R. Redox environment of the cell as viewed through the redox
state of the glutathione disulfide/glutathione couple. Free radical biology & medicine
30, 1191-1212 (2001).

Serviddio, G., et al. Evidence of lower oxidative stress in the air spaces of patients with
reversible COPD. International journal of immunopathology and pharmacology 19,
617-628 (2006).

Slebos, D.J., et al. Mitochondrial Localization and Function of Heme Oxygenase-1 in
Cigarette Smoke-Induced Cell Death. Am J Respir Cell Mol Biol (2006).

Stevenson, C.S., Koch, L.G. & Britton, S.L. Aerobic capacity, oxidant stress, and chronic
obstructive pulmonary disease--a new take on an old hypothesis. Pharmacology &
therapeutics 110, 71-82 (2006).

Taylor, E.R., et al. Reversible glutathionylation of complex I increases mitochondrial
superoxide formation. The Journal of biological chemistry 278, 19603-19610 (2003).

Toorn van der , M., et al. Cigarette smoke induced blockade of the mitochondrial
respiratory chain switches lung epithelial cell apoptosis into necrosis. American
journal of physiology (2007).

Turrens, J.F. & Boveris, A. Generation of superoxide anion by the NADH dehydrogenase of
bovine heart mitochondria. Biochem J 191, 421-427 (1980).

Velsor L, Kariya C, Kachadourian R, and Day B, Mitochondrial Oxidative Stress in the
Lungs of Cystic Fibrosis Transmembrane Conductance Regulator Protein Mutant
Mice, American Journal of Respiratory Cell and Molecular Biology. Vol. 35, pp.
579-586, 2006

Wamelink, M. M., Struys, E. A., and Jakobs, C. (2008) The biochemistry, metabolism and
inherited defects of the pentose phosphate pathway: a review. J. Inherit. Metab. Dis.
31, 703–717.

Wu M., A. Neilson, A.L. Swift, R. Moran, J. Tamagnine, D. Parslow, S. Armistead, K.
Lemire, J. Orrell, J. Teich, S. Chomicz and D.A. Ferrick, Multiparameter metabolic
analysis reveals a close link between attenuated mitochondrial bioenergetic function
and enhanced glycolysis dependency in human tumour cells, Am. J. Physiol. Cell
Physiol. 292 (2007), pp. C125–C136.

56

Yang Se-Ran, Jessica Wright, Mark Bauter, Kathryn Seweryniak, Aruna Kode and Irfan
Rahman, Sirtuin regulates cigarette smoke-induced proinflammatory mediator release
via RelA/p65 NF-B in macrophages in vitro and in rat lungs in vivo: implications for
chronic inflammation and aging, Am J Physiol Lung Cell Mol Physiol, 292:567-576,
2007.

Yang Se-Ran, Asiya S. Chida, Mark R. Bauter, Nusrat Shafiq, Kathryn Seweryniak, Sanjay
B. Maggirwar, Iain Kilty and Irfan Rahman, Cigarette smoke induces
proinflammatory cytokine release by activation of NF-B and posttranslational
modifications of histone deacetylase in macrophages, Am J Physiol Lung Cell Mol
Physiol 291: L46–L57, 2006

Yoshida T, Tuder R M, 2007, Pathobiology of cigarette smoke-induced chronic obstructive
pulmonary disease. Physiol Rev 87: 1047–1082.

Zhong, C.Y., Zhou, Y.M., Joad, J.P. & Pinkerton, K.E. Environmental tobacco smoke
suppresses nuclear factor-kappaB signaling to increase apoptosis in infant
monkey lungs. American journal of respiratory and critical care medicine 174, 428-
436 (2006).

3R4F research grade cigarette’s preliminary analysis available at
http://www.ca.uky.edu/refcig/3R4F%20Preliminary%20Analysis.pdf

Abstract (if available)

Abstract Chronic obstructive pulmonary disease (COPD) is a persistent obstruction of airways in the lungs due to the narrowing of the passage ways. Cigarette smoking is responsible for approximately 80-90% of COPD-related deaths. Cigarette smoke contains over 4000 chemicals, including carcinogens, strong electrophiles and other toxins. The current study was undertaken to determine the role of cigarette smoke in the development of mitochondrial dysfunction due to impairment of the energy-redox axis. Rat lung epithelial cell cultures (RLE-6TN cell line) and male A/J mice were used to study the effects of cigarette smoke extract (CSE) or cigarette smoke (CS), respectively. The effects of individual constituents of CSE like acrolein and nitric oxide on cell viability and redox status of the cell was also assessed. We have adapted a method to prepare cigarette smoke extract in our laboratory by smoking 4 cigarettes in 20 ml of serum free F-12 media. Our data indicate that treatment of RLE-6TN cells with doses less than 2.5% CSE for 4 h, and whole body smoke exposure for 2 weeks in A/J mice lead to an increase in the NADPH/NADP+ ratio with no change in the NADH/NAD+ ratio as measured by HPLC. At 24 h, a decrease in both NADPH/NADP+ and NADH/NAD+ ratios, indicate of an initial compensatory response to cigarette smoke. This increase in NADPH was found to be because of the increased activity of the pentose phosphate pathway. The other redox couple of GSH/GSSG was also shown to increase.

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Asset Metadata

Creator Agarwal, Amit (author)

Core Title Effect of cigarette smoke on redox regulation in chronic obstructive pulmonary disease

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 08/09/2010

Defense Date 07/10/2010

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cigarette smoke,OAI-PMH Harvest,redox

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Cadenas, Enrique (committee chair), Okamoto, Curtis Toshio (committee member), Shen, Wei-Chiang (committee member)

Creator Email amitagarwalusc@gmail.com,aragarwa@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3350

Unique identifier UC1474553

Identifier etd-Agarwal-3961 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-378889 (legacy record id),usctheses-m3350 (legacy record id)

Legacy Identifier etd-Agarwal-3961.pdf

Dmrecord 378889

Document Type Thesis

Rights Agarwal, Amit

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu