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: klkhanduja@rediffmail.com (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.

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