Life Sciences 76 (2005) 2669–2680
www.elsevier.com/locate/lifescie
Inhibitory effect of vitamin E on proinflammatory cytokines-and
endotoxin-induced nitric oxide release in alveolar macrophages
Krishan Lal KhandujaT, Pramod Kumar Avti, Surender Kumar,
Vandana Pathania, Chander Mohan Pathak
Department of Biophysics, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India
Received 4 June 2004; accepted 7 September 2004
Abstract
Nitric oxide is thought to be an important modulator of various functions in normal and inflamed airways. In
the present study, we evaluated the effects of high vitamin E (250 mg and 1250 mg a-tocopheryl acetate (TA)/
kg diet/10 days) on nitric oxide (NOS) release by alveolar macrophages (AMs) in response to lipopolysaccharide
(LPS), interleukin-1h (IL-1h) and tumor necrosis factor (TNF-a). LPS and IL-1h treatment (1-10 Ag/ml)
enhanced NOS release in AMs from control animals fed on 50 mg vitamin E/kg diet in a concentration
dependent manner. However, this enhancement of NOS was attenuated in the AMs of animals fed with 250 mg
or 1250 mg vitamin E/kg diet. TNF-a had no effect in eliciting the release of NOS in AMs obtained either from
control or from hyper vitamin E fed animals. Further, LPS (1-10 Ag/ml) enhanced the inducible nitric oxide
synthase (iNOS) activity of AMs of control group and TA-fed animals almost to equal extent. Similarly, LPS-
induced formation of N-nitrosamine (N-nitroso-L-[14C]-proline) in AMs of control and TA-supplemented
animals were not different statistically. On the other hand, in vitro addition of vitamin E (200 AM) in AMs of
control animals, when triggered with 10 Ag LPS/ml, caused a significant decrease in N-nitroso-L-[14C]-proline
formation. It seems that high doses of TA in diet may play a role in reducing the lipopolysaccharide and
proinflammatory cytokines-induced NOS-mediated damage by AMs.
D 2005 Published by Elsevier Inc.
Keywords: Alveolar macrophages; Vitamin E; Proinflammatory cytokines; LPS; iNOS; N-nitrosamines
0024-3205/$ -
doi:10.1016/j.l
T Correspond
E-mail add
see front matter D 2005 Published by Elsevier Inc.
fs.2004.09.037
ing author. Tel.: +91 172 2747585x5246; fax: +91 172 2744401.
ress: [email protected] (K.L. Khanduja).
K.L. Khanduja et al. / Life Sciences 76 (2005) 2669–26802670
Introduction
In lungs, alveolar macrophages (AMs) form the first line of defense against the invading foreign
material, and maintain the sterility of the lower respiratory tract. They help lungs to perform the vital
functions of gaseous exchange. The resident AMs down regulate the immune response and try to prevent
the generation of inflammatory responses (Thepen et al., 1992). However, with persistent exposure to
environmental insults, macrophages remain in the activated state and perform a myriad of functions e.g.
phagocytosis, free radical generation, release of mediators in the process of inflammation and cell
mediated immunity which leads to a variety of pulmonary inflammatory diseases (Calhoun et al., 1992).
The effect of various mediators such as TNF-a, IL-1h, IL-6, IL-8, prostaglandins, leukotrienes and
platelet activating factors on AMs is obscure (Park et al., 2001; Jussila et al., 2002; Careau et al., 2002).
Endotoxins like LPS, is known to activate macrophages and enhance the release of various mediators
(Lee et al., 2003). The secreted proinflammatory cytokines further activate macrophages in an autocrine
mechanism to produce increased levels of NOS(Srivastava et al., 2002).
NOS is synthesized from guanido nitrogen atom of L-arginine through the action of nitric oxide
synthase. NOS is lipophilic in nature and can easily cross membranes by simple diffusion (Thomas et
al., 2001). It is highly reactive, with a short half-life and gets easily oxidized to stable nitrite and
nitrate (Nathan, 2002). Nitric oxide has recently been recognized as an important messenger
molecule, having a broad spectrum of functions in many biological systems ranging from
physiological control to pathological cytotoxic effects (Nathan and Shiloh, 2000). In physiological
states, NOS plays a protective role, but under conditions of high output it may contribute to tissue
damage by reacting with superoxide to form peroxynitrite (Burgner et al., 1998; Gaut et al., 2002).
Recent studies show that myeloperoxidase and other peroxidases use H2O2 and NO2� to nitrate
tyrosine in vitro, indicating that peroxidases contribute to the generation of reactive nitrogen species
and inflammation in vivo. Various proinflammatory agents and endotoxins like TNF-a, IF-g, IL-1,
IL-2, LPS act on certain cells, induce iNOS activity (Xie et al., 1993) leading to the increased
production of nitric oxide. The cytotoxic effects of NOS have been attributed to the products such as
peroxynitrite, which results in the inhibition of cell replication by inactivation of enzymes such as
ribonucleotide reductase, and impairs mitochondrial respiration. NOS and its derivatives are not only
key molecules that damage DNA, but also modify the structure and function of proteins that are
involved in the maintenance of cellular homeostasis and integrity. Hence the measurements of
damaged products are useful for assessing the risk or for developing chemoprevention strategies in
nitrosative overload diseases. Some promising specific markers used in nitrosative stress include N-
nitrosoproline apart from others including N-nitrosoaminoacid and NO3�in plasma, 8-oxo-dG, 8-
nitrosoguanosine, exocyclic etheno and malondialdehyde-DNA adducts in target cells and 3-
nitrotyrosine products (Marnett and Plastarns, 2001). Although, considerable information is available
on the physiological role of NOS in the process of signal transduction, but, recently much attention
has been diverted towards its cytotoxic effects in various organs. NOS may act as a free radical in
various inflammatory diseases and may also lead to the production of carcinogenic nitrosamines.
Thus, overproduction of NOS by AMs might play a pivotal role in many pulmonary inflammatory
diseases such as asthma (Kharitonov et al., 1994), bronchiectasis (Kharitonov et al., 1995),
tuberculosis (Choi et al., 2002), sepsis (Kobayashi et al., 1998; Skidgel et al., 2002), cystic fibrosis
(Paredi et al., 1999), ARDS (Sittipunt et al., 2001) and chronic inflammation, which is a recognized
risk factor for the development of cancer.
K.L. Khanduja et al. / Life Sciences 76 (2005) 2669–2680 2671
For controlling the increased nitrosative burden, agents that are normally taken in the diet and have
scavenging effect might prove promising. One such agent is vitamin E, which apart from its role as an
antioxidant, is also known to have immunomodulatory effects. Though, numerous reports have shown
involvement of vitamin E in diverse functions but its role in proinflammatory cytokine and endotoxin-
induced NOS stress in AMs is still scarce. Therefore, the emphasis of the present study was to elucidate
the role of dietary supplementation of vitamin E on endotoxin and proinflammatory cytokine-induced
NOS and nitrosocompound formation in AMs.
Materials and methods
Materials
Nicotinamide adenine dinucleotide phosphate (NADPH), tumor necrosis factor-a (TNF-a),
lipopolysaccharide (LPS, from E.Coli serotype 055:B5), interleukin-1h, dl-a-tocopheryl acetate (a-
TA), TLC plates (polyester, silica gel, 250 Am layer thickness), N-monomethyl-L-arginine were
purchased from Sigma Chemicals Co., MO, USA. Fetal calf serum (FCS) was purchased from Hi-
Media, India. L-[U-14C]-proline (Sp.act. 250 mCi/mmol) and l-[U-14C]-arginine were purchased from
Board of Radioisotope Technology (BRIT), Mumbai, India. All other reagents of analytical grade were
purchased locally.
Methods
Vitamin E diet
Male Wistar rats (200–250 g) were fed with basal diet containing 20% vitamin free casein, 10%
sucrose, 57% starch, 8% vitamin E stripped sunflower oil, 4% mineral mixture and 1% vitamin
mixture. Stripping of vitamin E from the oil was done according to Mohri et al. (1983). Before
starting the experiment, all the animals were fed basal diet containing 50 mg vitamin E/kg diet
(control diet) for 3 days. The animals were divided into three groups of 6 rats each and were fed diets
containing 50, 250 and 1250 mg vitamin E/kg respectively. Food and water were given ad libitum for
10 days. Vitamin E in AMs was measured spectrofluorometrically according to the method of Taylor
et al. (1976).
Bronchoalveolar lavage collection, isolation and culture of alveolar macrophages (AMs)
After 10 days AMs were isolated according to the method of Myrvik et al. (1961). Briefly, the
trachea was cannulated and lungs were lavaged with a total volume of 50 ml PBS in 6–8 ml aliquots.
Lavage was centrifuged at 800�g for 10 minutes at 4 8C. The pellet obtained was subjected to
hypotonic shock with 0.16 M ammonium chloride in Tris-HCl buffer (pH-7.2) to remove contaminating
erythryocytes. This procedure was repeated and the pellet obtained was then washed twice with EarleTsbalanced salt solution (EBBS) and then suspended in RPMI-1640 medium containing 200 U/ml
penicillin, 100 Ag/ml streptomycin, 2.5 Ag/ml amphotericin B, 2 mM sodium bicarbonate, 50 AM 2-
mercaptoethanol and 50 mM HEPES buffer, pH-7.4. Cell viability was determined by 0.4% trypan blue
exclusion. More than 99% cells were found to be viable. On Wright staining, more than 95% cells were
found to be AMs.
K.L. Khanduja et al. / Life Sciences 76 (2005) 2669–26802672
Endotoxin and proinflammatory cytokine treatment
5.0�105 cells/ml/well were seeded in 24 well plate and incubated at 37 8C in 5% CO2 for 2 hrs. The
non-adherent cells were removed and the monolayer were further cultured for 48 hrs on treatment with
different concentrations of LPS, IL-1h and TNF-a.
Assessment of Nitric Oxide (NO) release
5.0�105 cells/ml/well were seeded in 24 well plate and incubated at 37 8C in 5% CO2 for 2 hrs the
non-adherent cells were removed and the monolayer were further cultured for 48 hrs on treatment with
different concentrations of LPS, IL-1h and TNF-a. Nitric oxide formation by AMs was assayed by
measuring the amount of nitrite (NO2�nmol/mg protein) formed in the medium, according to the method
of Tian and Lawrence (1995). Fifty microlitre of deproteinized supernatant (medium) were mixed with
equal volume of Greiss reagent in flat bottom 96 well plate. The plate was incubated at 37 8C for 10 min,
and then read at 540 nm by using an ELISA reader. A standard curve was drawn using sodium nitrite (5–
50 AM) solution in medium.
Inducible nitric oxide synthase (iNOS) assay
Activity of iNOS was determined by measuring the conversion of radiolabeled L-arginine to L-
citrulline according to the method of Salter et al. (1991). Briefly, the reaction mixture contained 1 mM
dithiothreitol, 1 mM EGTA, 0.1 mM NADPH, 300 AM BH4, 1.55 AM [14C]-arginine in 100 Al of 30mM HEPES buffer, pH-7.4 in test as well as reference sample taken in 10 ml polypropylene tubes.
Cell lysate was added to both the tubes so that the final volume did not exceed 200 Al. Tubes were
incubated at 37 8C for 30 min in water bath. Then 1.5 ml of 1:1 (v/v) double distilled water: Dowex-
50W (200-400, 8% cross linked, Na+-form) was added per tube and vortexed for 2 min and allowed to
settle for 10 min. Two microlitre of supernatant containing [14C]-citrulline was removed and put in 10
ml scintillation fluid. Counting was done in a liquid scintillation counter (LKB WALLACE, Finland).
The disintegration per minute (DPM) values obtained were subtracted from those obtained by
including 1 mM LN G-Monomethylarginine, acetate salt (NMMA),a NOS inhibitor, in the reaction
mixture. This gave the Ca2+-independent NOS (iNOS) activity in macrophages. The enzyme activity
was expressed as nmol [14C]-citrulline formed/30 min/mg protein.
Estimation of N-Nitrosoproline formation by AMs
The formation of N-nitroso-compounds was measured by estimating the N-nitrosation of L-[14C]-
proline according to the method of Young et al. (1977) by ascending thin layer chromatography with
slight modification. AMs obtained from vitamin E supplemented animals were treated with LPS (1
and 10 Ag/ml) in the medium containing 15 mM cold proline and 1.0 ACi/ml [14C]-proline for 48
hrs. Contents of each well were transferred into eppendorf tubes and centrifuged at 800�g for 10
min at 37 8C. One millilitre each of supernatant and 5N NaOH was mixed to stop the reaction
followed by N-nitrosoproline extraction with 1 ml of dichloromethane. The contents of the tubes
were vortexed for 2 min. and then centrifuged at 3000 rpm for 10 min. An aliquot (0.6 ml) of this
lower (dichloromethane) layer was taken and evaporated and redissolved in 50 Al dichloromethane.
This was then applied on polyester-silica gel TLC-plate, activated at 110 8C for 1 hr prior to use.
TLC plates were developed in acetonitrile:chloroform:95% ethanol:acetic acid (100:100:97:3) and
then dried. TLC plates was cut 1 cm wide strips and put successively into 10 ml scintillation fluid
after scrapping. For each sample Rf value for N-nitroso-L-[14C]-proline formation was similar to that
K.L. Khanduja et al. / Life Sciences 76 (2005) 2669–2680 2673
of standard (N-nitroso-L-[14C]-proline) prepared by a direct in vitro reaction between L-[14C]-proline
and sodium nitrite as described by Braunberg and Dailey (1973) with slight modification. In short, to
1 ml of 0.07% sodium nitrite solution, 2 ACi [14C]-proline was added. The mixture was incubated at
room temperature for 4 hrs in dark. Then 1 ml of reaction mixture was treated with 1 ml of 5 N
NaOH to stop reaction. N-Nitroso-L-[14C]-proline was extracted with 1 ml dichloromethane. 600 Alof extract was evaporated under nitrogen and redissolved in 50 Al dichloromethane and then applied
on TLC plate as described above. The material scrapped from each centimeter length of TLC plate
was counted successively and a graph was plotted to know the Rf value of N-Nitroso-L-[14C]-
proline. To study the effect of in vitro addition of vitamin E (0–200 AM) on N-nitrosoproline
formation by AMs, the study was repeated after obtaining AMs from the control animals as described
earlier in the text.
Results
Release of nitric oxide by AMs in response to LPS, IL-1b and TNF-a
We studied the effect of vitamin E supplementation on NOS release from AMs of rats treated with
varying concentrations of LPS (0.1, 1 and 10 Ag/ml) in vitro Fig. 1. In AMs from the control group,
0.1 Ag LPS/ml in the culture media did not induce NOS production and was similar to that of
unstimulated levels in the respective groups. However, incubation of AMs with LPS at 1 and 10 Ag
0
50
100
150
200
250
300
350
400
50 250 1250Vitamin E (mg/kg diet)
Nit
ric
oxi
de
form
atio
n (
nm
ol/m
g p
rote
in)
0 µg LPS/ml0.1 µg LPS/ml1 µg LPS/ml10 µg LPS/ml
* **
*
µµ
µµ
Fig. 1. Effect of dietary supplementation of different doses of vitamin E to rats on nitric oxide release by rat alveolar
macrophages treated with varying concentrations of LPS. Values mean F SD; (n = 6), * p b 0.01 compared with groups fed
with 50 mg vitamin E/kg diet.
0
50
100
150
200
250
300
50 250 1250Vitamin E (mg/kg diet)
Nit
ric
oxi
de
form
atio
n (
nm
ol/m
g p
rote
in) 0 ng IL-1β/ml
1 ng IL-1β/ml10 ng IL-1β/ml100 ng IL-1β/ml
* *
Fig. 2. Effect of dietary supplementation of different doses of vitamin E to rats on nitric oxide release by rat alveolar
macrophages treated with different concentrations of IL-1h. Values mean F SD; (n = 6), * p b 0.05 compared with groups fed
with 50 mg vitamin E/kg diet.
K.L. Khanduja et al. / Life Sciences 76 (2005) 2669–26802674
LPS with AMs retrieved from animals 10 days after feeding vitamin E at a dose of 250 mg and 1250
mg/kg diet could induce NOS formation slightly which was not significantly different as compared to
their respective control groups. However, the induction of NOS by LPS (1 and 10 Ag/ml) was
0
20
40
60
80
100
120
140
160
180
200
50 250 1250
Vitamin E (mg/kg diet)
Nit
ric
oxi
de
form
atio
n (
nm
ol/m
g p
rote
in)
0 ng TNF-α /ml5 ng TNF-α /ml50 ng TNF-α /ml
Fig 3. Effect of dietary supplementation of different doses of vitamin E to rats on nitric oxide release by rat alveolar
macrophages treated with varying concentrations of TNF-a. Values mean F SD; (n = 6), No significant change was
observed in different vitamin E fed groups.
Table 1
Effect of different doses of vitamin E feeding to rats for 10 days on vitamin E content, and on iNOS activity in rat alveolar
macrophages incubated with 1 and 10 Ag LPS/ml
Vitamin E
(mg/kg diet)
Vitamin E content in
AMs (nmol/mg protein)
iNOS activity (nmol l-[14C]-citrulline formed/30
min/mg protein)
1 Ag LPS/ml 10 Ag LPS/ml
50 144 F 15.1 14.2 F 0.87 20.6 F 5.30
250 226 F 10.1T 16.7 F 3.48 22.1 F 4.48
1250 253 F 9.84T, a 13.6 F 2.17 16.8 F 4.0
Basal level of iNOS activity was 5.91 F 0.92 nmol 1-[14C]-citrulline formed/30 min/mg protein.
Values are mean F SD; (n = 6).
No significant change in iNOS activity was observed in different vitamin E fed groups.a p b 0.001, compared to 250 mg/kg diet group.
T p b 0.001, compared to 50 mg/kg diet group.
K.L. Khanduja et al. / Life Sciences 76 (2005) 2669–2680 2675
significantly (p b 0.01) inhibited in the AMs obtained from high vitamin E supplemented groups as
compared to control diet groups. The effect of vitamin E supplementation on NOS formation in
AMs by varying concentrations of IL-1h (1, 10 and 100 ng/ml) is shown in Fig. 2. In the control
group, treatment with 1, 10 and 100 ng IL-1h/ml increased the NOS production by 32%, 49% and
51% respectively. In AMs of animals pretreated with 250 mg and 1250 mg vitamin E/kg diet, IL-
1h/ml could not reduce NOS. Incubation of TNF-a at varying concentrations (5–50 ng/ml) did not
affect the NOS release in AMs of control and the animals pretreated with high dietary vitamin E
and the values in all the groups were found to be almost same as that of basal level (135 F 22
nml/mg protein) Fig. 3.
0
1
2
3
4
5
6
50 250 1250Vitamin E (mg/kg diet)
N-n
itro
so-L
-[14
C]-
Pro
line
form
atio
n(n
mo
l/mg
pro
tein
)
0 µg LPS/ml1 µg LPS/ml10 µg LPS/ml
µµµ
Fig 4. Effect of dietary supplementation of different doses of vitamin E to rats on N-nitroso-L-[14C]-proline formation by rat
alveolar macrophages treated with different concentrations of LPS. Values mean F SD; (n = 6), No significant change was
observed in different vitamin E fed groups.
Table 2
Effect of different concentrations of vitamin E in vitro on N-nitroso-L-[14C]-proline formation triggered by 10 Ag LPS/ml in rat
alveolar macrophages
Vitamin E Conc. (AM) N-nitroso-L-[14C]-proline
formation (nmol/mg protein)
0 4.20 F 0.51
50 4.425 F 0.91
100 3.49 F 0.70
200 2.33 F 0.40T
Values are mean F SD; (n = 6).
T p b 0.01, values compared with 0 AM vitamin E treatment.
K.L. Khanduja et al. / Life Sciences 76 (2005) 2669–26802676
Effect of dietary supplementation of vitamin E at high doses on inducible nitric oxide synthase (iNOS)
activity in AMs
Vitamin E content in the isolated AMs from 250 and 1250 mg vitamin E/kg diet fed groups increased
by 56% and 75% as compared to control. Since there was suppression in LPS-induced NOS release in
AMs due to vitamin E pretreatment (Fig. 1), further investigations on the activity of iNOS enzyme in
AMs was performed (Table 1). In control group, LPS at 0.1 Ag/ml concentration did not enhance iNOS
activity. However, treatment with 1 and 10 Ag LPS/ml enhanced the iNOS activity by 2.4 and 3.49 fold
respectively. Further, in high vitamin E fed animals, LPS did not cause any change of iNOS in AMs.
Effect of dietary supplementation of vitamin E at high doses on N-nitroso-L-proline formation by AMs
The formation of N-nitrosoproline by alveolar macrophages on stimulation with LPS is shown in Fig.
4. In the control diet group, treatment with 1 and 10 Ag LPS/ml increased the N-nitrosoproline formation
by 2.18 and 2.06 fold respectively. Dietary supplementation with 250 mg and 1250 mg vitamin E/kg diet
did not alter N-nitrosoproline formation. In vitro incubation of AMs of control diet with vitamin E at 100
and 200 AM concentration reduced the nitrosoproline formation by 17% and 45% respectively on
challenge with 10 Ag LPS/ml for 48 hours (Table 2).
Discussion
In the present study, we observed that LPS and IL-1h treatment significantly enhanced NOS release
from AMs in the control group. However, dietary supplementation of vitamin E for 10 days attenuated
the LPS-and IL-1h-induced nitric oxide production by AMs. Surprisingly, challenge with TNF-a did not
induce NOS release in the AMs. The inhibitory response in the induction of NOS production by vitamin
enriched AMs on treatment with both LPS and IL-1h may be due to scavenging of NOS by vitamin E.
The possible mechanism by which vitamin E acts as an antioxidant is electron transfer, in which the
radical cation is first formed followed and reversible deprotonation in solution. In some cases, other
factors may also play a role such as presence of bulky groups near the OH group (Pedulli et al., 1997),
hydrogen bonding characteristics of solvent (Barclay et al., 1999) or in a biological context, solubility
and transport to specific tissues (Burton et al., 1998). Also, a-tocopherol is located in the membrane with
the polar chromanol group near the surface and the hydrophobic tail extending towards the center of the
K.L. Khanduja et al. / Life Sciences 76 (2005) 2669–2680 2677
bilayer. The hydroxyl group at the 6-position in ring structure is optimal for scavenging free radicals and
terminating lipid peroxidation (Niki et al., 1985). Earlier, vitamin E has been suggested to play a role in
protection against nitrogen oxides, and has also been reported to inhibit peroxynitrite (formed by
reaction of O2- and NOS) induced phospholipid oxidation under in vitro conditions (Christen et al., 1997).
Recent evidence suggests that vitamin E oxidation is prevented by NOS in the lipophilic membrane
environment and prevents its loss. This combination together inhibits the lipid peroxidation process
thereby acting as lipid soluble chain breaking antioxidant (Rubbo et al., 2000). In our earlier studies we
have shown that vitamin E suppresses the superoxide formation (Pathania et al., 1999) and thus reduces
the formation of peroxynitrite. Thus alteration in the oxidant-antioxidant balance results in pathological
states. Studies on hypoxia-reoxygenation in cultured myocytes have shown that vitamin E alone or in
combination with other tocopherols results in the suppression of hypoxia-reoxygenation (Chen et al.,
2002). Free radicals, such as NOS, derived from normal physiological and metabolic processes of the
macrophages are essential for a cell because they act as signaling molecules (Baran et al., 2004). On the
other hand their excessive production has detrimental effect on the lung tissue. Therefore, it is quite
essential to maintain the cellular homeostasis, as the products of NOS metabolites are known to act as
potent oxidizing agents. In the present study, inhibition of LPS-and proinflammatory cytokines-induced
NOS in AMs by vitamin E suggests the reduction in damage to the surrounding lung tissue.
One of the pathways for NOS induction could be due to the enhanced expression of iNOS. In
macrophages, iNOS has been reported to be expressed after several hours of exposure to cytokines like
IL-1h and/or microbial products such as a bacterial LPS (Tabakman et al., 1998). We observed no
change in LPS-induced iNOS activity in AMs of high vitamin E supplemented rats. This may be due to
the fact that different forms of protein kinases C play an important role in the induction of iNOS in
macrophages (Chio et al., 2004). Protein kinase C independent signal transduction pathways have also
been suggested to regulate the induction and activity of iNOS in rat AMs (Tabakman et al., 1998). Since
vitamin E has been reported to suppress protein kinase C activity in vascular smooth muscle cells (Azzi,
2004), there could be some other mechanisms in AMs, which might be influencing iNOS and affecting
NOS production, in addition to the free radical scavenging potential (Li et al., 1999). Apart, from this
vitamin E is known to play an important role in phosphorylation of nitric oxide synthase and help in
attenuation of platelet aggregation and lipid peroxidation (Li et al., 2001). In this regard, involvement of
transcription factors like NF-nB, as the potential target cannot be denied, since iNOS induction occurs is
via activation of transcription factor NF-nB (Xie et al., 1994). Moreover, NF-nB controls the expression
of a wide variety of gene activation in inflammation that includes cytokines (IL-1h and TNF-a),
enzymes like iNOS and cyclooxygenase-2 (COX-2), adhesion molecules and acute phase proteins
(Kleemann et al., 2003). Recently it was shown that vitamin E inhibits the nitric oxide induced COX-2
activity in macrophages (Beharka et al., 2002). Therefore, the presence of vitamin E in high amounts
might be altering the activation of AMs via blocking the NF-nB activation, which may explain its
anticancer and anti-inflammatory effects. However, this needs further experimental verification.
Activated macrophages can produce potentially carcinogenic nitrosamines, such as the N-nitro-
socompounds. Since activated AMs produced enhanced levels of NOS on stimulation with endotoxin and
proinflammatory cytokines, the formation of nitrosamines by AMs cannot be ruled out. In the present
study, we investigated the effect of high dietary intake of vitamin E on N-nitrosoproline (an index for the
formation of N-nitroso-compounds) formation by AMs in response to endotoxin and IL-1h treatment.
Although, these agents increased the N-nitrosoproline formation in the control group, there was no
alteration in LPS-induced N-nitrosoproline formation by AMs in high vitamin E supplemented groups.
K.L. Khanduja et al. / Life Sciences 76 (2005) 2669–26802678
Interestingly, in vitro studies revealed that, high vitamin E at 200 AM concentration caused a significant
decrease in N-nitrosoproline formation by AMs reflecting protective role of vitamin E during the
inflammatory responses. Therefore, it seems that by supplementing even 1250 mg vitamin E/kg diet to
the animals, vitamin E in AMs did not reach to the optimum level that is required to inhibit N-nitrosation
of proline as was observed in in vitro studies. Moreover, the nitrogen metabolites produced by the AMs
have the potential to damage their own components. Therefore, all types of NOS mediated toxicity in
target cells also occur in the producer macrophage cells.
Conclusion
In conclusion, vitamin E supplementation may be useful in reducing the risk of free radical mediated
damage to AMs themselves and also to the surrounding tissue during nitrosative stress and inflammatory
conditions.
References
Azzi, A., 2004. The role of alpha-tocopherol in preventing disease. European Journal Nutrition 43 (1), 118–125.
Baran, C.P., Zeigler, M.M., Tridandapani, S., Marsh, C.B., 2004. The role of ROS and RNS in regulating life and death of blood
monocytes. Current Pharmaceutical Design 10 (8), 855–866.
Barclay, L.R.C., Edwards, C.E., Vinqvist, M.R.J., 1999. Media effects on antioxidant activities of phenols and catechols.
Journal of American Chemical Society 121, 6226–6231.
Beharka, A.A., Wu, D., Serafini, M., Meydani, S.N., 2002. Mechanism of vitamin E inhibition of cyclooxygenase activity in
macrophages from old mice: role of peroxynitrite. Free Radical in Biology and Medicine 32, 503–511.
Burgner, D., Rockett, K., Kwiatkowski, D., 1998. Nitric oxide and infectious diseases. Archieves Disease in Childhood 81,
185–188.
Braunberg, R.C., Dailey, R.E., 1973. Formation of nitrosoproline in rats. Proceedings in Society of Experimental Biology and
Medicine 142, 993–996.
Burton, G.W., Traber, M.G., Acuff, R.V., Walters, D.N., Kayden, H., Hughes, L., Ingold, K.U., 1998. Human plasma and tissue
alpha-tocopherol concentrations in response to supplementation with deuterated natural and synthetic vitamin E. American
Journal of Clinical Nutrition 67 (4), 669–684.
Calhoun, W.J., Reed, H.E., Moest, D.R., Stevens, C.A., 1992. Enhanced superoxide production by alveolar macrophages and
air-space cells, airway inflammation and alveolar macrophages density changes after segmental antigen bronchoprovocation
in allergic subjects. American Reviews in Respiratory Disorder 145, 317–325.
Careau, E., Sirosis, J., Bissonnette, E.Y., 2002. Characterization of lung hyperresponsiveness, inflammation and alveolar
macrophage mediator production in allergy resistant and susceptible rats. American Journal of Respiratory Cell and
Molecular Biology 26 (5), 579–586.
Chen, H., Li, D., Saldeen, T., Romeo, F., Mehta, J.L., 2002. Mixed tocopherol preparation is superior to alpha-tocopherol alone
against hypoxia-reoxygenation injury. Biochemistry Biophysics Research Communication 291, 349–353.
Chio, C.C., Chang, Y.H., Hsu, Y.W., chi, K.H., Lin, W.W., 2004. PKA-dependent activation of PKC, p38 MAPK and IKK in
macrophage: implication in the induction of inducible nitric oxide synthase and interleukin-6 by dibutyryl camp. Cell
Signaling 16 (5), 565–575.
Choi, H.S., Rai, P.R., Chu, H.W., Cool, C., Chan, E.D., 2002. Analysis of nitric oxide synthase and nitrotyrosine in human
pulmonary tuberculosis. American Journal of Respiratory Critical Care Medicine 166 (2), 176–186.
Christen, H.S., Woodall, A.A., Shigenaga, M.K., Southwell-Kelly, P.T., Duncan, M.W., Ames, B.N., 1997. g-Tocopherol traps
mutagenic electrophiles such as NOX and complements a-tocopherol: Physiological implications. Proceedings of National
Academy of Science (U. S. A.) 94, 3217–3222.
K.L. Khanduja et al. / Life Sciences 76 (2005) 2669–2680 2679
Gaut, J.P., Byun, J., Tran, H.D., Lauber, W.M., Carroll, J.A.A., Hotchkiss, R.S., Belaouaj, A., Heinecke, J.W., 2002.
Myeloperoxidase produces nitrating oxidants in vivo. Journal of Clinical Investigations 109, 1311–1319.
Jussila, J., Kamulainen, H., Huttunen, K., Prponen, M., Livanainen, E., Torkko, P., Kosma, V.M., Perkonen, J., Hirronen, M.R.,
2002. Mycobacterium terrace isolated from indoor air of a moisture-damaged building induces sustained biphasic
inflammatory response in mouse lungs. Environmental Health Perspectives 110 (11), 1119–1125.
Kharitonov, S.A., Wells, A.U., O’Lonnor, B.J., et al., 1995. Elevated levels of exhaled nitric oxide in bronchiectasis. American
Journal of Respiratory Critical Care Medicine 151, 1894–1899.
Kharitonov, S.A., Yates, D.H., Robins, R.A., et al., 1994. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 343,
133–135.
Kleemann, R., Gervois, P.P., Verschuren, L., Staels, B., Princen, H.M., Kooistra, T., 2003. Fibrates down-regulate IL-1-
stimulated C-reactive protein gene expression in hepatocytes by reducing nuclear p50-Nfkappa B-C/EBP-beta complex
formation. Blood 101 (2), 545–551.
Kobayashi, A., Hashimoto, S., Kooguchi, K., Kitamura, Y., Onodera, H., Urata, Y., Ashihara, T., 1998. Expression of
inducible nitric oxide synthase and inflammatory cytokines in alveolar macrophages of ARDS following sepsis. Chest
113, 1632–1639.
Lee, D.U., Kang, Y.J., Park, M.K., Lee, Y.S., Seo, H.G., Kim, T.S., Kim, C.H., Chang, K.C., 2003. Effects of 13-alkyl-
substituted berberine alkaloids on the expression of COX-II, TNF-alpha, iNOS and IL-12 production in LPS-stimulated
macrophages. Life Science 73 (11), 1401–1412.
Li, D., Saldeen, T., Romeo, F., Mehta, J.L., 1999. Relative effects of alpha-and gamma-tocopherol on low-density lipoprotein
oxidation and superoxide dismutase and nitric oxide synthase activity and protein expression in rats. Journal of
Cardiovascular Pharmacology and Therapeutics 4, 219–226.
Li, D., Saldeen, T., Romeo, F., Mehta, J.L., 2001. Different isoforms of tocopherols enhance nitric oxide synthase
phosphorylation and inhibit human platelet aggregation and lipid peroxidation: implications in therapy with vitamin E.
Journal of Cardiovascular Pharmacology and Therapeutics 6, 155–161.
Marnett, L.J., Plastarns, J.P., 2001. Endogenous DNA damage and mutation. Trends in Genetics 17, 214–221.
Mohri, K., Dohomoto, C., Idesu, H., Igarashi, O., 1983. A simple elimination method of vitamin E from vegetables and fish oil
for the preparation of vitamin E deficient diet. Journal of Japanese Society Nutrition Food Science 36, 122–124.
Myrvik, Q.N., Leake, E.S., Fariss, B., 1961. Study on pulmonary alveolar macrophages from the normal rabbit. A technique to
procure them in a high state of purity. Journal of Immunology 86, 128–132.
Nathan, C., 2002. Points of control in inflammation. Nature 420, 846–852.
Nathan, C., Shiloh, M.U., 2000. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and
microbial pathogens. Proceedings of National Academy of Science (U. S. A.) 97, 8841–8848.
Niki, E., Kawakami, A., Saito, M., Yamammoto, Y., Tsuchiya, J., Kamiya, Y., 1985. Effect of phytyl side chain of vitamin E on
its antioxidant activity. Journal of Biological Chemistry 260, 2191–2196.
Paredi, P., Shah, P.L., Montuschi, P., Sullivan, P., Hodson, M.E., Kharitonov, S.A., Barnes, P.J., 1999. Increased carbon
monoxide in exhaled air of patients with cystic fibrosis. Thorax 54, 917–920.
Park, W.Y., Goodman, R.B., Steinberg, K.P., Ruzinski, J.T., Radella, F., Park, D.R., Pugin, J., Skerrett, S.J., Hudson, L.D.,
Martin, J.R., 2001. Cytokine balance in the lungs of patients with acute respiratory distress syndrome. American Journal of
Respiratory Critical Care Medicine 164, 1896–1903.
Pathania, V., Syal, N., Pathak, C.M., Khanduja, K.L., 1999. Vitamin E suppresses the induction of reactive oxygen species
release by lipopolysaccharide, interleukin-1h and tumor necrosis factor-a in rat alveolar macrophages. Journal of Nutritional
Science and Vitaminology 45, 675–686.
Pedulli, G.F., Lucarini, M., Pedrielli, P., 1997. Free radicals in biology and environment. In: Minisci, F. (Eds.), Academic
Publishers, Dordrecht, The Netherlands, p. 169.
Rubbo, H., Radi, R., Anselmi, D., Kirk, M., Barnes, S., Butler, J., Eiserich, J.P., Freeman, B.A., 2000. Nitric oxide reaction with
peroxyl radicals spares alpha-tocopherol during lipid peroxidation. Greater oxidant protection from the pair nitric oxide/
alpha-tocopherol than alpha-tocopherol/ascorbate. Journal of Biological Chemistry 275, 10812–10818.
Salter, M., Knowler, R.G., Moncada, S., 1991. Wide spread tissue distribution, species distribution and changes in activity of
Ca2+-dependent and Ca2+-independent nitric oxide synthases. Federation European Biochemical Societies 291, 145–149.
Sittipunt, C., Sternberg, K.P., Ruzinski, J.T., Myles, C., Zhu, S., Goodman, R.B., Hudson, L.D., Matalon, S., Martin, T.R.,
2001. Nitric oxide and nitrotyrosine in the lungs of patients with acute respiratory distress syndrome. American Journal of
Respiratory Critical Care Medicine 163, 503–510.
K.L. Khanduja et al. / Life Sciences 76 (2005) 2669–26802680
Skidgel, R.A., Gao, X., Brovkoych, V., Rahman, A., Jho, D., Predescu, S., Standiford, T.J., Malik, A.B., 2002. Nitric oxide
stimulated macrophage inflammatory protein-2 expression in sepsis. Journal of Immunology 169, 2093–2101.
Srivastava, A.D., Rom, W.N., Jagirdar, J., Yie, T., Gordon, T., Tchou-wong, K., 2002. Crucial role of interleukin-1h and nitric
oxide synthase in silica-induced inflammation and apoptosis in mice. American Journal of Respiratory Critical Care
Medicine 165, 527–533.
Tabakman, R., Lazarovia, P., Matsuda, Y., Brodie, C., Oradia, H., 1998. Protein kinase D-independent selective induction of
nitric oxide synthase activity in rat alveolar macrophages by staurosporine. Nitric oxide: biology and chemistry. Supplement
Archives of Biochemistry and Biophysics 2, 50–58.
Taylor, S.L., Lamden, M.P., Tappel, A.L., 1976. Sensitive fluorometric method for tissue tocopherol analysis. Lipids 11,
530–538.
Thepen, T., Hoeben, K., Breve, J., Kraal, G., 1992. Alveolar macrophages down regulate local pulmonary immune responses
against intratracheally administered T-cell dependent, but not T-cell-independent antigens. Immunology 76, 60–64.
Thomas, D.D., Liu, X., Kantrow, S.P., Lancaster Jr., J.R., 2001. The biological lifetime of nitric oxide: implications for the
perivascular dynamics of NO and O2. Proceedings of National Academy of Science (U. S. A.) 98 (1), 355–360.
Tian, L., Lawrence, D.A., 1995. Lead inhibits nitric oxide production in vitro by murine splenic macrophages. Toxicology and
Applied Pharmacology 132, 156–163.
Xie, Q.W., Kashiwatara, Y., Nathan, C., 1994. Role of transcription factor NF-nB /Rel in induction of nitric oxide synthase.
Journal of Biological Chemistry 269, 4705–4708.
Xie, Q.W., Whisnant, R., Nathan, C., 1993. Promoter of mouse gene encoding calcium-independent nitric oxide synthase
confers inducibility by interferon gamma and bacterial lipopolysaccharide. Journal of Experimental Medicine 177,
1779–1784.
Young, J.C., Khan, S.U., Marriage, P.B., 1977. Fluorescent detection and determination of glyphosate via its N-nitroso
derivation by thin layer chromatography. Journal of Agriculture Food Chemistry 25, 918–922.
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