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International Agency for Research on Cancer, F-69372 Lyon Cedex 08, France [B. P., C-Q. L., P. B., H. O.]; The Stokes Research Institute and Department of Pediatrics, Neonatology Division, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19140 [Q. C., H. I.]; Bremen Institute for Prevention Research and Social Medicine (BIPS), 28199 Bremen, Germany [W. A.]; Institute of Environmental Medicine, Karolinska Institute, 17177 Stockholm, Sweden [F. N.]; Institute of Carcinogenesis, 115478 Moscow, Russia [A. M.]; National Research Center for Environment and Health (GSF), 85758 Munich, Germany [I. B-H.]; Regional Epidemiological Unit, 00198 Rome, Italy [C. F.]; and National Institute of Public Health, 76256 Bucharest, Romania [V. C.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Proteins are major targets for oxidative and nitrative damage in
vivo (16, 17, 18)
. Exposure of proteins to reactive
oxygen or nitrogen species results in modification of amino acid
residues, altering the protein structure and function (17
, 18)
. Free and protein-bound
NTYR,4
a stable product of nitration of tyrosine residues, has been measured
as a biomarker of protein damage induced by peroxynitrite and other
reactive nitrogen species (18)
. Increased levels of free
NTYR in the plasma of cigarette smokers and of protein-bound NTYR in
bronchoalveolar lavage fluids from patients with acute respiratory
distress syndrome have been reported (19, 20, 21)
. Moreover, a
recent study identified ceruloplasmin, transferrin,
1-antichymotrypsin, 1-protease inhibitor, and the ß-chain of
fibrinogen as the major nitrated plasma proteins in patients with acute
respiratory distress syndrome (21)
. Similarly, carbonyl
groups in proteins, determined as DNPH derivatives, have been analyzed
as a biomarker of oxidative damage of proteins (17)
.
Increased carbonyl levels in peripheral blood globin of smokers,
compared with nonsmokers, have been reported (22)
.
Modified forms of proteins accumulate during aging, oxidative stress,
and some pathological conditions (17
, 18) .
To bridge the gap between laboratory experiments and the population-based epidemiological study, the present transitional study (23) was performed. We have used immunodot blot and WB assays to determine levels of both nitrated and oxidized proteins in human plasma samples from cases of lung cancer and controls stratified according to cigarette smoking and exposure to ETS. We found that levels of nitrated proteins are significantly elevated in plasma samples from lung cancer patients compared with healthy controls, and that levels of oxidized protein are higher in smokers than nonsmokers. Furthermore, we identified some of these modified proteins present in human plasma using immunoprecipitation and WB analyses with specific antibodies to human plasma proteins.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cases and controls were administered a validated and standardized questionnaire (25 , 26) that included detailed sections on active smoking, ETS exposure during childhood, ETS exposure during adulthood from the spouse and at the workplace, occupational exposures, and family history of cancer. The section on ETS exposure had been used in a previous study conducted in Europe (25) and previously validated against urinary cotinine measurements (26) .
We defined as nonsmokers those cases and controls who had smoked less than 400 cigarettes, or the equivalent amount of tobacco as cigars, cigarillos or pipe tobacco, during their life time. This corresponds to about one cigarette per day for 1 year. Nonsmokers were classified as exposed or unexposed to ETS from the spouse or at the workplace. Smokers were classified according to cumulative consumption of tobacco, expressed as pack-years. Two groups of smokers were formed, one of heavy smokers (range 21–143 pack-years) and one of light smokers (range 0.3–4.3 pack-years). Ex-smokers were defined as smokers who had stopped smoking for at least 1 year before the start of the study.
We included 52 lung cancer cases and 43 controls, of whom 24 were from
the general population and 19 from hospitals. Eighty-five % of the
subjects were enrolled at six centers in Sweden, Germany, Italy,
Romania, and Russia. Table 1
reports selected characteristics of study subjects. Cases and controls
were similar with respect to sex, age, and tobacco consumption.
However, there were more women among nonsmokers than among smokers
(P of the difference, 0.001). Histopathological types of
cancer include squamous cell carcinoma (15 cases, of whom 4 were
nonsmokers), small cell carcinoma (8 cases, of whom 4 were nonsmokers),
adenocarcinoma (21 cases, of whom 17 were nonsmokers), and other and
mixed types (8 cases, of whom 3 were nonsmokers). Informed consent was
obtained from all of the patients and the protocol was approved by the
local and IARC Ethical Committees.
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Preparation of Standard Nitrated and Oxidized Human Plasma
Proteins.
Human plasma proteins (albumin, transferrin, and fibrinogen; Sigma, St.
Louis, MO) were incubated at 1 mg/ml with 1 mM
peroxynitrite in 0.5 M phosphate buffer (pH 7.4).
Peroxynitrite was synthesized in a quenched flow reactor, and excess
hydrogen peroxide was destroyed by granular manganese dioxide
(27)
. Peroxynitrite-treated transferrin (1 mg/ml) was
incubated for 16 h at 50°C with 1 mg of dialyzed Pronase E.
Concentrations of NTYR were measured by high-performance liquid
chromatography using a postseparation on-line reduction column
and an electrochemical detector, as reported previously
(28)
. The concentration of protein carbonyls in human
fibrinogen oxidized by peroxynitrite was determined
spectrophotometrically after formation of the DNPH derivative, as
previously described (29)
. Peroxynitrite-treated
transferrin and fibrinogen, which contained 8 nmol of NTYR/mg of
protein and 400 nmol of carbonyls/mg of protein, respectively, were
used as standards.
Preparation of DNPH-derivatized Proteins for SDS-gel
Electrophoresis and Immunodot Blot Analysis.
DNPH was prepared as a 20-mM solution in 10%
trifluoroacetic acid (30
, 31)
. A 20-µg aliquot of plasma
proteins was placed in a 1.5-ml tube, dried under vacuum, and then
resuspended in 3 µl of H2O. The sample was
mixed with 3 µl of 12% SDS and 6 µl of the DNPH solution and
incubated for 20 min at room temperature. The solution was neutralized
by addition of 4.5 µl of 2 M Tris base/30% glycerol
(v/v), giving final concentrations of 0.54 M and 8.2%
(v/v), respectively. The derivatized and neutralized samples were
applied to 7.5% SDS PAGE or dot-blotted onto a membrane.
Immunodot Blot Analysis of NTYR and Oxidized Proteins.
Twenty µg of plasma proteins were used either directly for NTYR
analysis or after DNPH-derivatization for analysis of oxidized proteins
using previously published methods (32
, 33) that were
modified as follows: proteins were diluted in 200 µl of PBS and
dotted onto a Millipore Immobilon-P membrane using a Bio-Dot SF
microfiltration apparatus (Bio-Rad). The membrane was blocked using 5%
nonfat dried milk for 1 h at room temperature and incubated with
either a mouse anti-NTYR monoclonal IgG (Upstate Biotechnology, Lake
Placid, NY) at a dilution of 1:2000 or a rabbit anti-DNP antibody
(Oxyblot; Oncor, Gaithersburg, MD) at a dilution of 1:300 overnight at
4°C, followed by either a goat antimouse secondary antibody
conjugated to horseradish peroxidase (1:4000 dilution) or a horseradish
peroxidase-conjugated goat antirabbit secondary antibody (1:400) for
2 h at room temperature. The membrane was then examined by
chemiluminescence using a Covalab kit (Dako, Trappes, France) and
exposed to hyperfilm (Amersham Pharmacia Biotech, Orsay, France), and
the films were exposed for 3 min. Transferrin and fibrinogen treated
with peroxynitrite were used as standards for NTYR and oxidized
proteins, respectively. Blot densities were measured by scanning the
films using a GS-670 Imaging Densitometer (Bio-Rad). Concentrations of
nitrated and oxidized plasma proteins were calculated using calibration
curves drawn with different concentrations of standard proteins (see
typical examples in Fig. 1
). The variation between duplicate measurements was 10–15%. Detection
limits for the analyses of nitrated and oxidized proteins were 0.08 and
0.5 nmol/mg protein, respectively.
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Identification of Nitrated and Oxidized Plasma Proteins by
Immunoprecipitation and WB Analyses.
An aliquot of 125 µg plasma proteins was used to perform
immunoprecipitation experiments for identification of the nitrated
proteins as described previously (21)
. The sample volume
was adjusted to 500 µl with immunoprecipitation buffer [20
mM Tris-base (pH 7.4), 140 mM NaCl, 10%
glycerol, 1% Triton X-100, 4 mM EGTA, supplemented with 10
µg/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride in
methanol] and precleaned by incubation with 15 µl of protein G
(Amersham Pharmacia Biotech) at 4°C for 1 h. After
centrifugation at 10,000 x g for 3 min, the
supernatant was transferred to a new tube and incubated with 2 µl (1
µg/µl) of mouse anti-NTYR monoclonal antibody overnight at 4°C,
followed by incubation with 30 µl of protein G at 4°C for 1 h.
After centrifugation at 10,000 x g for 2
min, the pellet was washed with 0.5 ml of immunoprecipitation buffer.
This procedure was repeated four times. After the last centrifugation,
the pellet was resuspended in 30 µl of 2.5x Laemmli buffer, heated
for 8 min at 100°C, electrophoresed through a 12% SDS-PAGE, and
transferred to Millipore Immobilon-P membrane. The membrane was blocked
for 2 h at room temperature with 10% nonfat dried milk and
incubated with one of the following seven rabbit polyclonal antibodies
against human plasma proteins: ferritin (1:8,000 dilution), fibrinogen
(1:4,000 dilution), albumin (1:5,000 dilution), plasminogen (1:5,000
dilution), transferrin (1:5,000 dilution), ceruloplasmin (1:5,000
dilution), antichymotrypsin (1:5,000 dilution); (all of the above
protein antibodies from Dako), and one mouse monoclonal antibody
against human 1-antitrypsin (1:5,000 dilution;
Calbiochem, La Jolla, CA). The membrane was further incubated with a
secondary goat antirabbit (1:3,000 dilution) or antimouse (1:3,000
dilution) antibody conjugated to horseradish peroxidase (Dako). The
membrane was then observed by supersignal ultrachemiluminescence
(Pierce, Rockford, IL).
Similarly, an aliquot of 125 µg plasma protein was derivatized with DNPH, and the DNPH-derivatized proteins were immunoprecipitated with 3 µl (0.5 µg/µl) of rabbit anti-DNP antibody and examined by WB analyses using the above antibodies to identify oxidized plasma proteins.
The results from immunoprecipitation and WB analyses were confirmed with reverse immunoprecipitation experiments. The plasma proteins were first immunoprecipitated with one of the above-mentioned antihuman plasma protein antibodies and then analyzed by WB analyses using either mouse anti-NTYR monoclonal antibody or rabbit anti-DNP antibody.
Plasma samples from healthy humans, which contained no detectable level of nitrated or oxidized proteins (<0.08 and <0.5 nmol/mg protein, respectively) were used as negative specimens. Several control experiments were also carried out, including immunoprecipitation without anti-NTYR or anti-DNP antibody, or WB analyses without any specific plasma protein antibodies. All of the experiments were repeated at least two or three times to confirm results.
Statistical Analyses.
We compared the mean levels of oxidized and nitrated proteins according
to smoking habits and according to case/control status, using
Student’s t test. In addition, we conducted a
multiple linear regression analysis of the association between levels
of oxidized and nitrated proteins and smoking habit and case/control
status, after controlling for potential confounding factors (sex, age,
and center). We also conducted a multiple logistic regression analysis,
again with adjustment for sex, age, and center; in this case, elevated
levels of oxidized and nitrated proteins were treated as the exposure
variables, and smoking habit and case/control status were the outcome
variables (35)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Human transferrin and fibrinogen
treated with peroxynitrite were used as standards to quantify nitrated
and oxidized proteins, respectively. Table 2
presents the levels of nitrated and oxidized proteins determined by
immunodot blot analyses in human plasma of lung cancer patients and
controls according to their smoking status. In positive plasma samples,
concentrations of nitrated proteins were in the range of 0.10–1.0 nmol
of NTYR/mg of protein, and those of oxidized proteins were from 1.0 to
8.0 nmol of carbonyls/mg protein.
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and Fig. 2, c and d
. Some plasma
samples contained up to five bands for NTYR-containing proteins, but
the number of bands varied between samples (Table 2)
, and many
NTYR-positive samples contained three major bands, with relative
molecular masses of Mr
205,000, 110,000, and 90,000 (Fig. 2c)
. Only one
carbonyl-containing protein band with a relative molecular mass of
Mr 60,000 was found (Fig. 2d)
. The levels of both nitrated and oxidized plasma
proteins determined on the basis of the number and intensity of bands
detected by WB analyses correlated well with those determined by
immunodot blot analyses.
Multivariate Statistical Analysis.
Table 3
presents the results of the multivariate linear regression analysis
based on WB analysis. After adjustment for potential confounders,
smoking status was associated with elevated levels of oxidized proteins
but not of nitrated proteins. No difference between heavy and light
smokers was seen for either type of protein modification. Among
nonsmokers, ETS exposure was not associated with elevated levels of
oxidized proteins and was inversely associated with the level of
nitrated proteins.
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. This difference, however, was present among smokers but not among
nonsmokers. Similar results on the effect of tobacco smoking, ETS exposure, and case/control status were obtained when the analysis was based on results of immunodot blot analysis rather than WB results (not shown in detail). Similarly, the results of the logistic regression analysis provided evidence of an association between smoking status and elevated levels of oxidized proteins (odds ratio for WB analysis, 4.0; 95% confidence interval, 1.4–11) and between case status and elevated levels of nitrated proteins (odds ratio for WB analysis 2.5; 95% confidence interval, 1.0–5.9).
Identification of Specific Nitrated and Oxidized Human Plasma
Proteins.
To identify nitrated and oxidized proteins present in human plasma from
lung cancer patients and smokers, we immunoprecipitated modified
proteins using anti-NTYR and anti-DNP antibodies. The
immunoprecipitated proteins were examined by WB analyses using eight
different antibodies specific for human plasma proteins, which were
selected on the basis of their abundance in human plasma and molecular
mass. Typical results are shown in Fig. 3a
for nitrated proteins. The plasma proteins
immunoprecipitated with the anti-NTYR antibody were analyzed by WB
using an array of antibodies against human proteins. When the
immunoprecipitates with the anti-NTYR antibody of human plasma, which
contained nitrated proteins initially, were analyzed by WB, bands were
observed at expected molecular weights with the following antibodies.
Antibody against human fibrinogen showed two bands at
Mr 55,000 and 65,000, which may
correspond to the and 
chains (Lane 1). Antibodies
against transferrin, plasminogen, and ceruloplasmin showed one band at
Mr 77,000 (Lane 3), one
band at Mr 95,000 (Lane 5)
and two bands at about Mr 140,000
(Lane 7), respectively. No bands were detected with
antibodies against human albumin, ferritin, antichymotrypsin, and
antitrypsin (data not shown). When a human plasma sample that did not
contain nitrated proteins initially was examined in a similar manner,
no bands corresponding to the specific proteins were detected by WB
analyses with any of the eight antibodies (Lanes 2,
4, 6, and 8 in Fig. 3a
).
|
, ß and -chains (Fig. 3b)
. No bands at expected molecular
weights were detected with the other antibodies. When a human plasma
sample which did not contain oxidized proteins initially was examined,
no bands corresponding to the specific proteins were detected by WB
analyses with any of the eight antibodies (Lanes 1,
7, and 8 in Fig. 3b
). Reverse immunoprecipitation experiments were carried out to confirm these results. The immunoprecipitates with antibodies against fibrinogen, transferrin, plasminogen, and ceruloplasmin showed the occurrence of nitrated proteins at expected molecular weights, which were detected with anti-NTYR antibody (data not shown). The immunoprecipitates of the DNPH-derivatized protein with antifibrinogen contained the protein, which was detected by anti-DNP antibody (data not shown). On the basis of these results, we conclude that the major nitrated proteins present in human plasma from lung cancer patients were fibrinogen, transferrin, plasminogen, and ceruloplasmin and that the oxidized protein found frequently in the plasma of smokers was fibrinogen.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our major findings in this study were (a) elevated levels of nitrated proteins in lung cancer patients compared with those in controls; and (b) elevated levels of oxidized proteins in smokers compared with nonsmokers. Plasma samples from patients with lung cancer, especially those of smokers, showed high levels of nitrated proteins. These results suggest that during development of lung cancer, production of reactive nitrogen species is increased and that this mechanism might be involved in, but not limited to, tobacco-related lung cancer development. It has been reported that activities of nitric oxide synthase and expression of inducible nitric oxide synthase were increased in patients with lung cancer (38, 39, 40, 41) and a variety of respiratory disorders (42 , 43) . In view of the expression of inducible nitric oxide synthase in various cancerous and precancerous tissues in humans, it would be interesting to study whether levels of nitrated proteins are elevated in plasma of patients with other types of cancer, and whether they could be measured as diagnostic markers for early detection of tumors.
Levels of oxidized protein were also higher in smokers than in nonsmokers. However, among nonsmokers, ETS exposure was not associated with increased levels of oxidized proteins. Increased oxidative stress in smokers has been shown by measuring various biomarkers, including lipid peroxidation products in plasma (6 , 7) , oxidized DNA bases in human leukocytes DNA and urine (11, 12, 13, 14 , 44) and F2-isoprostanes in plasma and urine (5 , 6) . It would be worthwhile to further study the correlation between these biomarkers and levels of oxidized proteins in relation to smoking habits.
Using immunoprecipitation and WB analyses, we identified fibrinogen, transferrin, plasminogen, and ceruloplasmin as major nitrated proteins present in plasma from lung cancer patients and fibrinogen as the only oxidized protein found frequently in the plasma of smokers. In contrast, a major human plasma protein, albumin, was neither nitrated nor oxidized. It has been reported that transition metals can catalyze peroxynitrite-mediated nitration of phenolic compounds (45) . Metalloproteins such as transferrin and ceruloplasmin can also interact with nitric oxide to form a metal-nitrosyl complex, which can generate nitrosonium cation (NO+), an electrophilic nitrosating agent (46 , 47) , which may react with tyrosine residues to form 3-nitrosotyrosine, which is then converted to NTYR by further oxidation (48) . Because levels of fibrinogen (49) and ceruloplasmin (50) are elevated in plasma of subjects with lung cancer, these plasma proteins could be better targets for nitration and oxidation when human plasma was exposed to oxidant(s) produced by the iron-ascorbic acid system, fibrinogen in plasma was much more susceptible to oxidative modification (carbonyl formation) than other major plasma proteins such as albumin, immunoglobulins, and transferrin (31) . Oxidized fibrinogen inhibits thrombin-catalyzed clot formation, whereas nitrated fibrinogen appears to accelerate this process (51 , 21) . Many other proteins and enzymes change their conformation and lose their function after modification of amino acid residues by reactive oxygen and nitrogen species (17 , 18) . Additional studies are needed to elucidate the biological significance of nitration and oxidation of human plasma proteins, especially fibrinogen, transferrin, plasminogen, and ceruloplasmin, in relation to coagulation, fibrinolysis, and metal ion metabolism, as well as to tumor growth, invasion, and metastasis.
Although proteins are major targets for oxidative and nitrative damage in vivo (16, 17, 18) , the modified proteins in human plasma have not until recently been extensively measured as possible biomarkers of oxidative stress in relation to human nutrition, disease status, life-style, and so forth. The immunoassays described in this study are sensitive and specific for oxidized and nitrated proteins. Only 20 µl of plasma is needed for the analyses. In addition, modified proteins could be measured as better biomarkers than modified DNA bases because (a) target proteins occur at higher concentrations than DNA; (b) they may exist for a longer period than modified DNA bases because they are possibly not repaired efficiently; and (c) during the analytical process described in this work, proteins may be less susceptible to artifactual modification than DNA bases. Additional studies are in progress in our laboratory using these methods, especially to investigate effects of antioxidants and cessation of cigarette smoking on levels of nitrated and oxidized proteins in human plasma.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported partially by a grant from the
Environment Programme of the European Commission, DG-XII (Contract
EV5V-CT94-0555). The work reported in this paper was undertaken during
the tenure of a Research Training Fellowship awarded by the IARC (to
C-Q. L.). This work was also supported by a Murray Fellowship from the
Joseph Stokes Jr. Research Institute (to Q. C.), Grants HL-59664 and
HL-60290 from NIH, and an Established Investigator Award from the
American Heart Association ( to H. I.). 
2 C-Q. L. is currently at the Massachusetts
Institute of Technology, Division of Bioengineering and Environmental
Health, Cambridge, MA 02139.
3 To whom requests for reprints should be
addressed, at the International Agency for Research on Cancer, 150
Cours Albert Thomas, 69372 Lyon, Cedex 08, France. Fax:
33-4-72-73-80-88; E-mail: ohshima{at}iarc.fr
4 The abbreviations used are: NTYR, nitrotyrosine;
DNP, dinitrophenyl; DNPH, 2,4-dinitrophenylhydrazine; ETS,
environmental tobacco smoke; WB, Western blot.
5 C-Q. Li, B. Pignatelli and H.
Ohshima, manuscript in preparation.
Received 5/15/00. Accepted 11/ 6/00.
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A. S. Haqqani, J. F. Kelly, and H. C. Birnboim Selective Nitration of Histone Tyrosine Residues in Vivo in Mutatect Tumors J. Biol. Chem., January 25, 2002; 277(5): 3614 - 3621. [Abstract] [Full Text] [PDF]
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