Therapeutic potential of arachidonyl trifluromethyl ketone, a cytosolic phospholipaseA2 IVA specific inhibitor, in cigarette smoke condensate-induced pathological conditions in alveolar type I & II epithelial cells.

Cigarette smoke is responsible for multiple disorders and causes almost 10 million annual deaths globally but underlying mechanisms are still underexplored. Continuous exposure of Cigarette smoke condensate (CSC) leads to cytosolic phospholipase A2 (cPLA2) mediated high free radicals where cPLA2s seems to play crucial role in generated various patho-physiological conditions such as chronic inflammation, oxidative stress and cancer. In this view, we assessed the therapeutic potential of arachidonyl trifluromethyl ketone (ATK), a cPLA2 inhibitor, via pharmacological inhibition of most expressible CSC-induced cPLA2 group IVA in type-I and type-II alveolar epithelial cells. The In Vitro inhibitory effect of ATK on CSC-induced PLA2 activity and its cellular role were assessed in terms of cell viability, fluorescein diacetate (FDA) dye uptake assay for membrane integrity, reactive oxygen species (ROS)/reactive nitrogen species (RNS) levels and pro apoptotic as well as anti apoptosis markers via flow cytometry, along with extracellular signal-regulated kinases (ERK) levels using enzyme-linked immunosorbent assay (ELISA). The experimental findings demonstrated that ATK acts as potent inhibitor of cPLA2 activity and shown its effectiveness as therapeutic agent by significantly mimicking CSC-induced levels of free radicals, primary apoptosis, ratio of pro-apoptotic/apoptotic proteins and levels of ERK whereas protected cells from loss of cell viability and membrane integrity. Thus, this study is an important step towards the opening up of avenues for the applicability of the cPLA2 isoform specific inhibitors such as ATK for pre-clinical and clinical studies and could be beneficial during smoking-induced lung pathological conditions.

Chemopreventive effect of artesunate in 1,2-dimethylhydrazine-induced rat colon carcinogenesis.

Artesunate (ART) is a semisynthetic derivative of artemisinin. Artemisinin and its derivatives have shown profound cytotoxicity and antitumor activity in addition to antimalarial activity in various studies. As the in vivo chemopreventive efficacy of ART in colon carcinogenesis has not been investigated so far, the aim of the current study was to study the chemopreventive effect of ART in 1,2-dimethylhydrazine (DMH)-induced rat colon carcinogenesis. Animals were divided into four groups (n = 6): Group I - vehicle (1 mM ethylenediaminetetraacetic acid), Group II - DMH (20 mg/kg), Group III - DMH + 5-fluorouracil (81 mg/kg), Group IV - DMH + ART (6.7 mg/kg). After completion of 15 weeks of treatment, rats were sacrificed under ether anesthesia by cervical dislocation for assessment of lipid peroxidation (LPO), antioxidant status, average number of aberrant crypt foci (ACF), and cytokine levels. ART administration significantly decreased the average number of ACF/microscopic field. Similarly, LPO level was decreased and antioxidant activities were enhanced after ART treatment. ART decreased the levels of proinflammatory cytokines and induced apoptosis in the colons of DMH-treated rats. The results of this study suggest that ART has a beneficial effect against chemically induced colonic preneoplastic progression in rats.

Increased synthesis of folate transporters regulates folate transport in conditions of ethanol exposure and folate deficiency.

Excessive alcohol consumption and dietary folate inadequacy are the main contributors leading to folate deficiency (FD). The present study was planned to study regulation of folate transport in conditions of FD and ethanol exposure in human embryonic kidney cell line. Also, the reversible nature of effects mediated by ethanol exposure and FD was determined by folate repletion and ethanol removal. For ethanol treatment, HEK293 cells were grown in medium containing 100 mM ethanol, and after treatment, one group of cells was shifted on medium that was free from ethanol. For FD treatment, cells were grown in folate-deficient medium followed by shifting of one group of cells on folate containing medium. FD as well as ethanol exposure resulted in an increase in folate uptake which was due to an increase in expression of folate transporters, i.e., reduced folate carrier, proton-coupled folate transporter, and folate receptor, both at the mRNA and protein level. The effects mediated by ethanol exposure and FD were reversible on removal of treatment. Promoter region methylation of folate transporters remained unaffected after FD and ethanol exposure. As far as transcription rate of folate transporters is concerned, an increase in rate of synthesis was observed in both ethanol exposure and FD conditions. Additionally, mRNA life of folate transporters was observed to be reduced by FD. An increased expression of folate transporters under ethanol exposure and FD conditions can be attributed to enhanced rate of synthesis of folate transporters.

Mechanistic insights of intestinal absorption and renal conservation of folate in chronic alcoholism.

Folate mediated one-carbon metabolism is of fundamental importance for various cellular processes, including DNA synthesis and methylation of biological molecules. Due to the exogenous requirement of folate in mammals, there exists a well developed epithelial folate transport system for regulation of normal folate homeostasis. The intestinal and renal folate uptake is tightly and diversely regulated and disturbances in folate homeostasis like in alcoholism have pathological consequences. The study was sought to delineate the regulatory mechanism of folate uptake in intestine and reabsorption in renal tubular cells that could evaluate insights of malabsorption during alcoholism. The folate transporters PCFT and RFC were found to be associated with lipid rafts of membrane surfaces in intestine and kidney. Importantly, the observed lower intestinal and renal folate uptake was associated with decreased levels of folate transporter viz. PCFT and RFC in lipid rafts of intestinal and renal membrane surfaces. The decreased association of folate transporters in lipid rafts was associated with decreased protein and mRNA levels. In addition, immunohistochemical studies showed that alcoholic conditions deranged that localization of PCFT and RFC. These findings could explain the possible mechanistic insights that may result in folate malabsorption during alcoholism.

A new perspective on the quercetin paradox in male reproductive dysfunction.

Dietary bioflavonoids represent a large class of polyphenolic compounds found in most plants. A significant number of flavonoids are reported to have beneficial health effects. Quercetin is one such flavonoid which has been reported to possess strong antioxidant properties. However, as far as male reproduction and fertility are concerned, controversial reports exist in the literature highlighting the antioxidant as well as a prooxidant character of quercetin, leaving much to the researcher's speculation. The present review therefore, aimed at addressing this paradoxical behavior of quercetin by taking into account the in vitro and in vivo studies conducted till date regarding its role in the maintenance of male reproductive potential. From the detailed survey of the published data, it appears that the conflicting biological effects of quercetin might relate to its dose and the redox state of the cell. Thus, the cellular toxicity of quercetin metabolites might overshadow the beneficial effects of its supplementation in subjects having reproductive dysfunction coupled with elevated oxidative stress leading to the paradoxical behavior of the flavonoid.

Decreased activity of folate transporters in lipid rafts resulted in reduced hepatic folate uptake in chronic alcoholism in rats.

Folic acid is an essential nutrient that is required for one-carbon biosynthetic processes and for methylation of biomolecules. Deficiency of this micronutrient leads to disturbances in normal physiology of cell. Chronic alcoholism is well known to be associated with folate deficiency, which is due in part to folate malabsorption. The present study deals with the regulatory mechanisms of folate uptake in liver during chronic alcoholism. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20 % solution) orally for 3 months, and the molecular mechanisms of folate uptake were studied in liver. The characterization of the folate transport system in liver basolateral membrane (BLM) suggested it to be a carrier mediated and acidic pH dependent, with the major involvement of proton coupled folate transporter and folate binding protein in the uptake. The folate transporters were found to be associated with lipid raft microdomain of liver BLM. Moreover, ethanol ingestion decreased the folate transport by altering the Vmax of folate transport process and downregulated the expression of folate transporters in lipid rafts. The decreased transporter levels were associated with reduced protein and mRNA levels of these transporters in liver. The deranged folate uptake together with reduced folate transporter levels in lipid rafts resulted in reduced folate levels in liver and thereby to its reduced levels in serum of ethanol-fed rats. The chronic ethanol ingestion led to decreased folate uptake in liver, which was associated with the decreased number of transporter molecules in the lipid rafts that can be ascribed to the reduced synthesis of these transporters.

Folate malabsorption is associated with down-regulation of folate transporter expression and function at colon basolateral membrane in rats.

Folates, an essential component (important B vitamin) in the human diet, are involved in many metabolic pathways, mainly in carbon transfer reactions such as purine and pyrimidine biosynthesis and amino acid interconversions. Deficiency of this micronutrient leads to the disruption of folate-dependent metabolic pathways that lead to the development of clinical abnormalities ranging from anaemia to growth retardation. Folate deficiency due to alcohol ingestion is quite common, primarily due to malabsorption. The present study dealt with the mechanistic insights of folate malabsorption in colonic basolateral membrane (BLM). Wistar rats (n 12) were fed 1 g/kg body weight per d ethanol (20 %) solution orally for 3 months and folate transport was studied in the isolated colonic BLM. The folate exit across colon BLM shows characteristics of carrier-mediated process with the major involvement of reduced folate carrier (RFC). The chronic ethanol ingestion decreased the uptake by decreasing the affinity by 46 % (P < 0·01) and the number of transport molecules by 43 % (P < 0·001) at the colon BLM. The decreased uptake was associated with down-regulation of proton-coupled folate transporter (PCFT) and RFC expression at mRNA and protein levels. The extent of decrease was 44 % (P < 0·01) and 24 % (P < 0·05) for PCFT and 23 % (P < 0·01) and 57 % (P < 0·01) for RFC at mRNA and protein levels, respectively. Moreover, folate transporters were associated with lipid rafts (LR) of colon BLM, and chronic alcoholism decreased the association of these transporters with LR.

Curcumin sensitizes lung adenocarcinoma cells to apoptosis via intracellular redox status mediated pathway.

The present study demonstrates that curcumin acts as pro-oxidant and sensitizes human lung adenocarcinoma epithelial cells (A549) to apoptosis via intracellular redox status mediated pathway. Results indicated that curcumin induced cell toxicity (light microscopy and MTT assay) and apoptosis (AnnexinV-FITC/PI labeling and caspase-3 activity) in these cells. These events seem to be mediated through generation of reactive oxygen species (ROS) and superoxide radicals (SOR) and enhanced levels of lipid peroxidation. These changes were accompanied by increase in oxidized glutathione (GSSG), reduced glutathione (GSH) and gamma-glutamylcysteine synthetase (gamma-GCS) activity, but decrease in GSH/GSSG ratio. The induction of apoptosis and decrease in GSH/GSSG ratio was also accompanied by sustained phosphorylation and activation of p38 mitogen activated protein kinase (MAPK). On the other hand, addition of N-acetyl cysteine (NAC), an antioxidant, blocked the curcumin-induced ROS production and rescued malignant cells from curcumin-induced apoptosis through caspase-3 deactivation. However, L-buthionine sulfoximine (BSO), a GSH synthesis blocking agent, further enhanced curcumin-induced ROS production and apoptosis in A549 cells. Decreased GSH/GSSG ratio seems to be a crucial factor for the activation of MAPK signaling cascade by curcumin. The study therefore, provides an insight into the molecular mechanism involved in sensitization of lung adenocarcinoma cells to apoptosis by curcumin.

Bacopa monniera Attenuates Scopolamine-Induced Impairment of Spatial Memory in Mice.

Scopolamine, an anticholinergic, is an attractive amnesic agent for discerning the action of candidate antiamnesic drugs. Bacopa monniera Linn (Syn. Brahmi) is one such antiamnesic agent that is frequently used in the ancient Indian medical system. We have earlier reported the reversal of diazepam-induced amnesia with B. monniera. In this study we wanted to test if scopolamine-induced impairment of spatial memory can also be ameliorated by B. monniera using water maze mouse model. The objective of study was to study the effect of B. monniera on scopolamine-induced amnesia. We employed Morris water maze scale to test the amnesic effect of scopolamine and its reversal by B. monniera. Rotarod test was conducted to screen muscle coordination activity of mice. Scopolamine significantly impaired the acquisition and retrieval of memory producing both anterograde and retrograde amnesia. Bacopa monniera extract was able to reverse both anterograde and retrograde amnesia. We propose that B. monniera's effects on cholinergic system may be helpful for developing alternative therapeutic approaches for the treatment of Alzheimer's disease.

Corticosteroids affect nitric oxide generation, total free radicals production, and nitric oxide synthase activity in monocytes of asthmatic patients.

Airways inflammation, a pathological hallmark of asthma, is associated with the recruitment of pro-inflammatory and inflammatory cells like eosinophils, polymorphonuclear leucocytes cells, mononuclear cells, macrophages, epithelial desquamation, and airways remodeling with sub-epithelial fibrosis. Activated inflammatory cells along with the resident cells can generate pro-inflammatory mediators including oxidants such as superoxide radicals, reactive oxygen species (ROS), and reactive nitrogen species. One of such inflammatory mediator that has received considerable attention is the nitric oxide (NO(•)) generated by pulmonary macrophageal/epithelial cells. In this study, we have explored that systemic monocytes also get activated in asthma to produce oxidants like ROS and NO(•). We estimated the NO(•) production, nitric oxide synthase (NOS) activity, inducible NOS (iNOS) mRNA levels and total free radical activity (TFRA) in blood monocytes of healthy control subjects, untreated asthmatic patients, patients on corticosteroid for less than 6 months and patients on corticosteroid for more than 6 months. Increase in NOS activity, NO(•) levels, and TFRA was observed in monocytes of asthmatic patients. The increase was found to be associated with the transcriptional upregulation of iNOS gene and severity of disease. Highest values of NOS activity, NO(•), and iNOS mRNA were found in the patients with acute asthma. Corticosteroid administration was found to be effective in reversing the induction of iNOS mRNA levels, NOS activity and NO(•) levels. Corticosteroids controlled asthma appears to have association with NOS, NO(•), and TFRA in systemic monocytes of the patients.

Correlation of seizures and biochemical parameters of oxidative stress in experimentally induced inflammatory rat models.

The role of oxidative stress in the pathogenesis of various conditions including epilepsy, inflammatory bowel disease and rheumatoid arthritis is evolving. The aim of this study was to find out the correlation between various inflammatory models with seizures and antioxidant parameters. Fifty-four male rats were divided into three groups of colitis, adjuvant arthritis and cotton wool granuloma (CWG). Each group had three subgroups of control, model and treatment. Thalidomide was used as treatment in colitis and arthritis group, whereas etoricoxib was used in CWG group. In colitis and arthritis groups, thalidomide was administered for 3 and 17 days, respectively, whereas etoricoxib was administered for 7 days in CWG group. At the end of treatment protocols, a subconvulsive dose of pentylenetetrazole (PTZ) (40 mg/kg i.p.) was injected intraperitoneally to note seizure onset and score. After confirming the presence of inflammation by morphological and histological studies, plasma and brain biochemical parameters of oxidative stress were estimated. The models of colitis, arthritis and CWG were effectively produced as evidenced by morphological scores (P < 0.001). Thalidomide reduced the morphological score (P < 0.002) and seizure grade (P < 0.001), whereas increased seizure onset (P < 0.001) in the arthritis group. There was an increase in malondialdehyde levels in the brain of thalidomide-treated groups (P < 0.002) and a significant decrease in superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels. There was neither improvement in seizure nor any significant changes in lipid peroxidation and antioxidant enzyme levels in etoricoxib-treated group. Thalidomide was effective in reducing the extent of arthritis as well as reducing the seizure scoring and increasing seizure onset in the adjuvant arthritis group. As it increased lipid peroxidation and reduced SOD and GPx, further evaluation is necessary with respect to oxidative stress.

Cigarette smoke condensate promotes cell proliferation through disturbance in cellular redox homeostasis of transformed lung epithelial type-II cells.

Present study was initiated to evaluate the effects of cigarette smoke condensate (CSC) on the cellular changes at molecular levels in non-small lung carcinoma cells (A549). Cigarette smoke condensate at low concentration (0.1 microg/ml) induced cancer cell proliferation, DNA synthesis, reduced glutathione (GSH) levels and intercellular adhesion molecule-1 (ICAM-1) expression without any significant change in reactive oxygen species (ROS) and superoxide radicals (SOR) production. The increased levels of GSH and ICAM-1 due to increased gamma-glutamylcysteine synthetase (gamma-GCS) activity and transcriptional activation of ICAM-1 gene respectively might be via activation of p38 mitogen activated protein kinase (p38 MAPK). The induction of ICAM-1 expression and cell proliferation reflect the tumor promoting activity of low CSC concentration. On the other hand, high CSC concentration (50 microg/ml), which is doubtful to be achieved in the lungs even in the chain smokers, induced killing effects on cancer cells by increasing apoptosis, ROS and SOR production, inducing cell cycle arrest, and increased ICAM-1 levels. These changes were found to be associated with altered GSH/GSSG ratio which shifted the redox balance towards more oxidizing equivalent followed by activation of p38 MAPK and stress-activated protein kinase (SAPK) involved in signaling cascade and finally transcriptional activation of gamma-GCS and ICAM-1 genes. These changes were found to be p38 and SAPK dependent.

ATRA promotes alpha tocopherol succinate-induced apoptosis in freshly isolated leukemic cells from chronic myeloid leukemic patients.

We investigated the in vitro efficacy of all-trans retinoic acid (ATRA) and alpha-tocopherol succinate (alpha-TS) alone and in combination on the induction of cell death in freshly isolated leukemic cells obtained from chronic myeloid leukemia (CML) patients. In vitro cytotoxicity and induction of lipid peroxidation by ATRA (10 microM) and alpha-TS (25 or 50 microM) were evaluated in primary leukemic cells by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and malondialdehyde formation respectively. Treatment of leukemic cells with alpha-TS alone or in combination with ATRA significantly (P < 0.05) decreased the cell viability in a concentration and time dependent manner as compared to peripheral blood mononuclear cells obtained from normal healthy controls. Lipid peroxidation was enhanced by 98% (P < 0.05) on combined treatment of cells with ATRA (10 microM) and alpha-TS (50 microM). ATRA alone did not enhance the externalization of phosphatidyl serine as studied by annexin-V binding using fluorescence activated cell sorter analysis, whereas in combination with alpha-TS it increased to 400% at 12 h. The treatment of leukemic cells to combination of ATRA with alpha-TS significantly decreased (P < 0.05) mitochondrial membrane potential and enhanced lysosomal destabilization. The combination of these drugs also increased mitochondrial and cytosolic reactive oxygen species (ROS) production, nitric oxide levels, and caspase-3 activity significantly and caused DNA fragmentation at 24 h in a concentration dependent manner in the leukemic cells. Our data suggest that ATRA in combination with alpha-TS efficiently induces apoptosis in leukemic cells, which may be a useful therapeutic modality in CML patients.

Whole body exposure to low-dose gamma radiation promotes kidney antioxidant status in Balb/c mice.

We examined the effect of whole body low-dose gamma-irradiation on the status of the antioxidant defense system in the rodent kidneys at different time intervals. Young male Balb/c mice were exposed to whole body radiation from a (60)Co source at doses of 10, 25 and 50 cGy (48.78 cGy/min). Antioxidant status and lipid peroxidation were estimated in the kidneys at 4, 12 and 24 h after irradiation. Lipid peroxidation increased between 33% and 49% and reduced glutathione between 12% and 47% at 12 h at different radiation doses. Reduced glutathione level remained significantly (p < 0.05) elevated even at 24 h after irradiation to 25 cGy. Superoxide dismutase activity also increased by 37% at 12 h on exposure of animals to all the doses up to 50 cGy. Catalase activity increased significantly at 12 h on exposure to 10 cGy and 50 cGy. Interestingly, glutathione peroxidase activity increased by 31% at 4 h and subsequently returned to control levels at 24 h after exposure to 50 cGy. Glutathione reductase activity increased by 10-12% at 12 h after exposure to 25 cGy and 50 cGy. The results suggest that the whole body exposure of animals to gamma radiation stimulates the antioxidant defense system in the kidneys within 4 to 24 h after irradiation, at doses of 25 cGy and 50 cGy.

Anti-apoptotic activity of caffeic acid, ellagic acid and ferulic acid in normal human peripheral blood mononuclear cells: a Bcl-2 independent mechanism.

Polyphenols have been shown to induce apoptosis in a variety of tumor cells including leukemia both in vitro and in vivo. However, their action on normal human peripheral blood mononuclear cells (PBMCs) during oxidative stress remains to be explored. In this study, we have evaluated the anti-apoptotic and radical scavenging activities of dietary phenolics, namely caffeic acid (CA), ellagic acid (EA) and ferulic acid (FA). H2O2-induced apoptosis in normal human PBMCs was assayed by phosphotidylserine externalization, nucleosomal damage and DNA fragmentation. Incubation of PBMCs with 5 mM H2O2 led to increased Annexin-V binding to externalized phosphatidyl serine (PS), an event of pre-apoptotic stage of the cell. Peripheral blood mononuclear cells pretreated with phenolics could resist H2O2-induced apoptotic damage. Caffeic acid (60 and 120 microM) and EA (100 and 200 microM) caused no change in externalization of PS, whereas FA (100 and 200 microM) increased externalization of PS in PBMCs treated with H2O2. The effects of phenolics were abolished to a large extent by culturing the PBMCs for 24 h after washing the phenolics from the medium. Inhibitory activities of these phenolics on lipid peroxidation were in the order of EA

Smokeless tobacco impairs the antioxidant defense in liver, lung, and kidney of rats.

The present study was designed to evaluate the effects of long-term use of aqueous extract of gutkha (a form of smokeless tobacco) on the antioxidant defense status and histopathological changes in liver, lung, and kidney of male Wistar rats. Animals were orally administered aqueous extract of smokeless tobacco (AEST) at a low dose (96 mg/kg body weight per day) for 2 and 32 weeks, and at a high dose (960 mg/kg body weight per day) for 2 weeks. High-dose AEST for 2 weeks decreased the hepatic glutathione (GSH) and glutathione peroxidase (GPx), and increased lipid peroxidation (Lpx) by 17%, 19%, and 20%, respectively. Low-dose AEST for 32 weeks significantly decreased (p < 0.05) the antioxidant status in these organs. In liver, AEST decreased GSH levels and the activities of superoxide dismutase (SOD), catalase (CAT), and GPx by 34.6%, 29%, 17.1%, and 17.4%, respectively, but it increased Lpx by 64%. In kidney, GSH, SOD, CAT, and GPx were decreased by 26.6%, 23%, 33%, and 18%, respectively, with an increase of Lpx by 65%. AEST decreased the lung GSH, SOD, CAT, and GPx, and increased lung Lpx by 43%, 28.5%, 37%, 40%, and 24%, respectively. However, no change in the plasma levels of vitamins A, C, and E were observed with AEST treatment. Histopathological findings suggest that administration of AEST at the high dose for 2 weeks or at the low dose for 32 weeks could cause mild to moderate inflammation in liver and lungs. In conclusion, a decrease in the antioxidant defense system and long-term inflammation caused by smokeless tobacco may be risk factors for gutkha-induced pathogenesis.

Nimesulide inhibits lipopolysaccharide-induced production of superoxide anions and nitric oxide and iNOS expression in alveolar macrophages.

The study was designed to investigate the effect of nimesulide on lipopolysaccharide (LPS)-induced proinflammatory oxidants production by rat alveolar macrophages (AMs). Effects of LPS and nimesulide on antioxidant defense and the expression of inducible nitric oxide synthase (iNOS) were also studied. It was found that nimesulide could scavenge superoxide anions (O2*-), nitric oxide (NO*) and total oxidant burden induced by LPS in AMs in vitro. Approximately 850 nmoles of nimesulide had activity equivalent to one IU of superoxide dismutase (SOD). Further, to confirm the in vitro observation, Male Wistar rats were orally administered with nimesulide (9 mg/kg b. wt. twice daily) for one week followed by intratracheal instillation of 2 microg LPS to stimulate lung inflammation. AMs from bronchoalveolar lavage fluid were collected 18 h after instillation of LPS. Nimesulide pretreatment could inhibit O2*-, NO() and lipid peroxidation in AMs. Nimesulide also suppressed LPS-induced iNOS expression in AMs in vivo and in vitro. Nimesulide could also normalize LPS-induced changes in the levels of superoxide dismutase (SOD), glutathione reductase (GR) and reduced glutathione (GSH) in AMs. Inhibition in production of oxidants in LPS-challenged AMs by nimesulide could be one of the pathways for its anti-inflammatory action.

Inhibitory effect of vitamin E on proinflammatory cytokines-and endotoxin-induced nitric oxide release in alveolar macrophages.

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 alpha-tocopheryl acetate (TA)/kg diet/10 days) on nitric oxide (NO(.)) release by alveolar macrophages (AMs) in response to lipopolysaccharide (LPS), interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF-alpha). LPS and IL-1beta treatment (1-10 microg/ml) enhanced NO(.) release in AMs from control animals fed on 50 mg vitamin E/kg diet in a concentration dependent manner. However, this enhancement of NO(.) was attenuated in the AMs of animals fed with 250 mg or 1250 mg vitamin E/kg diet. TNF-alpha had no effect in eliciting the release of NO(.) in AMs obtained either from control or from hyper vitamin E fed animals. Further, LPS (1-10 microg/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-[(14)C]-proline) in AMs of control and TA-supplemented animals were not different statistically. On the other hand, in vitro addition of vitamin E (200 microM) in AMs of control animals, when triggered with 10 microg LPS/ml, caused a significant decrease in N-nitroso-L-[(14)C]-proline formation. It seems that high doses of TA in diet may play a role in reducing the lipopolysaccharide and proinflammatory cytokines-induced NO(.)-mediated damage by AMs.