Increased inducible nitric oxide synthase in lung carcinoma of smokers

Abstract

BACKGROUND.

Cigarette smoking is well known to play an important role in the development of lung cancer. Inducible nitric oxide synthase (iNOS) can either promote or inhibit cell proliferation and growth, which makes its role in the development of malignant tumors controversial. The relation between cigarette smoking and iNOS in human lung cancer is unknown.

METHODS.

The study examined the levels of iNOS/NO in nonsmall-cell lung cancer (NSCLC) tissues of smokers and nonsmokers and in NSCLC cells (NCI-H23) treated by 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-specific carcinogen.

RESULTS.

The level of iNOS/NO was significantly higher in lung cancer tissues of smokers than that of nonsmokers. Unlike iNOS/NO, the activity of caspase-3 was reduced in the former compared with the latter. The expression of the cleaved caspase-3 was deceased in NCI-H23 cells treated with S-Nitroso-N-acetylpenicillamine (SNAP), an NO donor, whereas treatment with NG-methyl-L-arginine (NMA), an NO inhibitor, caused an increase in cleaved caspase-3. Consistent with the change in caspase-3, SNAP treatment inhibited cell death induced by UCN01, a potent cell death-inducer. NMA treatment greatly enhanced the sensitivity of the cells to UCN01. Further, the cells treated by NNK showed an increase in iNOS protein, accompanied by an elevation of cell proliferation.

CONCLUSIONS.

The study demonstrates that cigarette smoking promotes the level of iNOS/NO but suppresses the activity of caspase-3, which may lead to the proliferation and growth of lung cancer cells. Cancer 2008. © 2007 American Cancer Society.

Lung cancer continues to be the leading cause of cancer death in both men and women.1 Extensive prospective epidemiologic data clearly establish cigarette smoking as the major cause of lung cancer.2, 3 The connection between smoking and smoking-related death in China has particularly aroused attention because its inhabitants account for 20% of the world population and smoke 30% of the world's cigarettes.4 The development of lung cancer is associated with smoking in about 85% to 90% of cases.5 It is obvious that carcinogens that cause lung cancer are associated with the use of tobacco products. However, how cigarette smoking is linked to molecules that play a role in the pathogenesis of lung cancer still remains unclear or inconsistent.

Inducible nitric oxide synthase (iNOS) is an enzyme responsible for the production of nitric oxide (NO). NO is an important bioregulatory molecule that mediates a variety of actions such as vasodilatation and apoptosis. It is now widely accepted that iNOS is expressed in response to several stresses, including inflammatory cytokines and bacterial endotoxin.6, 7 There are several publications describing the altered NO or iNOS in lung cancer.8–12 Masri et al8 showed that the metabolite of NO, nitrotyrosine, was increased in the tumor regions of nonsmall-cell lung carcinoma (NSCLC) relative to nontumor-bearing regions. However, they failed to detect the alteration in iNOS between lung tumor and tumor-free regions. A study by Liu et al9 suggested an increase in the NO level that was mainly produced by the alveolar macrophages rather than by the tumor cells. In the report by Fujimoto et al10 the activity of NOS was found in lung adenocarcinoma but not in other types of lung cancers. Ambs et al11 did not find an increase in NOS in NSCLC. Marrogi et al12 observed a high iNOS level in lung large-cell carcinoma and lung adenocarcinoma but not in squamous cell carcinoma. Therefore, it appears that most studies have observed an increase in NO or iNOS in lung cancer, but the data are not consistent in different types of lung cancer. Further, these studies did not analyze the relation between iNOS/NO and cigarette smoking in lung cancer.

A number of studies have demonstrated a link between cigarette smoking and the alteration of NO in several types of cells. The increased level of NO or NOS was found in exhaled breath condensate, lower respiratory tract, and lung parenchyma of experimental subjects or rats exposed to cigarette smoke.13–15 Smokeless tobacco extract also induced the production of NO in hamster cheek pouch cells.16 In contrast to the increased NO via smoking, several reports show that smoking damages the production of NO or the expression of NOS is decreased in airway compartments, lung epithelial cells, blood monocytes, and platelets.17–20 There are also reports demonstrating that smoking has no effect on NO production or NOS expression.21–23 Taken together, the results of cigarette smoking and the NO level are inconsistent either in in vivo or in vitro studies. Considering the potential role of NO in carcinogenesis24 and the causative relation between cigarette smoking and the development of lung cancer,2, 3 analysis of whether and how cigarette smoking is linked to NO or iNOS is deemed an important aspect in the development of lung cancer. To the best of our knowledge, there is no report comparing the level of NO or iNOS in lung cancer tissues of smokers and nonsmokers.

MATERIALS AND METHODS

Patient Population

Sixty lung tissue samples were obtained from patients with NSCLC who underwent surgical resection at the Prince of Wales Hospital. All tumor specimens were confirmed by histologic examinations. Detailed clinical information about these samples is listed in Table 1. The stage of disease (TNM) was determined according to the current UICC criteria after pathologic examination of primary tumor and regional lymph nodes. This work was approved by the Human Research Ethics Committee at the Chinese University of Hong Kong.

Table 1. Clinical Characteristics of Smokers and Nonsmokers With Lung Cancer

Clinical characteristics	Smokers	Nonsmokers	P
* Smokes at least 1 pack of cigarettes per day.
Sex
Women	4	5	>.05
Men	26	25	>.05
Age, y	64.0 ± 9.3 (43-76)	64.1 ± 11.3 (37-83)	>.05
Stage
I	15	15	>.05
II	7	6
III	7	6
IV	1	3
Histology
Adenocarcinoma	14	15	>.05
Large cell	3	1
Squamous cell	13	14	>.05
Tumor differentiation
Well	2	2
Moderate	19	18	>.05
Poor	9	10	>.05
Cigarette smoking (year)*	38.1 ± 13.2 (10-60)	0	<.01

Chemicals

Cell culture medium, Dulbecco modified Eagle medium (DMEM), and fetal calf serum were purchased from Life Technologies (Grand Island, NY). Anti-human iNOS, Bcl-xL, Bax, and actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif). Cleaved caspase-3 antibody was from Cell Signaling (Danvers, Mass). ABC reagent was from Vector Laboratories (Burlingame, Calif). The chemiluminescent detection kit (ECL system) was obtained from Amersham Pharmacia Biotech (Piscataway, NJ) and the Total NO Assay kit from R&D Systems (Minneapolis, Minn). The caspase-3 activity assay kit was from Chemicon (Temecula, Calif). 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was purchased from Chemsyn Science Laboratories (Lenexa, Kan). S-Nitroso-N-acetylpenicillamine (SNAP) was from Calbiochem (San Diego, Calif). All other chemicals were from Sigma-Aldrich (St. Louis, Mo) if not otherwise indicated.

Cell Culture

A human NSCLC cell line, NCI-H23, obtained from the American Type Culture Collection (Manassas, Va), was used in this study. The cells were grown in DMEM supplemented with 10% heat inactivated fetal bovine serum, 100 U/mL of penicillin, and 100 mg/mL of streptomycin in a humidified 5% CO₂ atmosphere at 37°C. The cells were separated using 0.05% trypsin/0.53 mM EDTA when they reached subconfluence.

Immunohistochemical Examination

The immunohistochemical examination was carried out as in our previous publication.25 Briefly, after sectioning and blocking the sections were incubated with a primary antibody overnight at 4°C, followed by a biotinylate-labeled IgG secondary antibody. Antigen staining was visualized by diaminobenzidine (DAB) substrate. Negative controls were prepared by replacing the primary antibody with phosphate-buffered saline (PBS). The intensity of the antigen staining was scored according to the standard described in Table 2.

Table 2. Scoring Standard for Antigen Staining

Grade	Scoring by tissue	Scoring by cell
0	None	None
1	Slight	Part of the cytoplasm or nucleus
2	Moderate	Whole cytoplasm or nucleus with reticular deposits
3	Strong	Reticular deposits in <½ of cytoplasm or nucleus
4	Extremely strong	Reticular deposits in >½ of cytoplasm or nucleus

Western Blot Analysis

Western blot was carried out as in our previous publication.25 Briefly, samples were homogenized at 4°C to isolate proteins. The proteins isolated were separated on 10% (v/v) sodium dodecyl sulfate (SDS)-polyacrylamide gels and electrophoretically transferred from the gel onto nitrocellulose membranes. The membrane was then incubated with a primary antibody, followed by a secondary antibody, IgG-HRP. Finally, the membrane was treated with the ECL reagents to observe the protein bands.

Assay for NO Level

The assay was carried out using a Total NO Assay kit from R&D Systems. The principle of the assay is to convert nitrate to nitrite. Nitrate and nitrite are the stable degradation products of NO. Thus, measurement of total nitrite can be used to reflect the level of NO.26, 27 Briefly, samples were homogenized at 4°C in a buffer containing 0.1 mM ethylene diamine tetra acetic acid (EDTA), 10 μg/mL leupetin, 10 μg/mL aprotinin, 10 μg/mL soybean trypsin inhibitor, 100 μm p-amidinophenymethanesulfonyl fluoride hydrochloride, 1 μM dithiothreitol, 0.32 M sucrose, and 15 mM hydroxyethylpiperazine-N′-2-ethanesulfonic acid. The homogenates were then centrifuged at 100,000g for 1 hour at 4°C to collect the supernatants for NO assay.

Caspase-3 Activity Measurement

The activity of caspase-3 was measured using a caspase-3 activity assay kit (Chemicon). The assay is based on the detection of the chromophore p-nitroaniline (pNA) after it is cleaved from the labeled substrate DEVD-pNA. Briefly, samples were lysed and incubated for 1 hour at 37°C with the caspase-specific substrate in the provided reaction buffer. Cleaving of Ac-DEVD-pNA by active caspase resulted in the liberation of pNA (p-nitroanilide) into solution. All experiments were repeated in triplicate.

DNA Fragmentation Measurement

DNA fragments in the cytoplasm were labeled with BrdU and the BrdU-labeled DNA fragment was then detected using an enzyme-linked immunosorbent assay (ELISA) kit from Roche (Mannheim, Germany). The assay was performed according to the manufacturer's instructions.

MTT Cell Proliferation Assay

The proliferation of lung cancer cells was assessed with 3-(4,5-dimethylthiazol-2-yi)-2,5-diphenyltetrazolium (MTT). The cells were plated at a density of 2000 cells/well in 96-well tissue culture dishes. After 3 cell washes with PBS, 10 μL MTT (5 mg/mL in PBS) was added. The cells were incubated for 4 hours at 37°C in a humidified atmosphere, 5% CO₂. The medium was aspirated and cells solubilized in 200 μL dimethylsulphoxide (DMSO). The absorbance at 570 nm was measured using a reference wavelength of 630 nm with a microplate reader. The proliferation of the cells was presented as a percent of control culture conditions.

Statistics

All values are expressed as mean ± standard error. The relations between nonparameter variables were analyzed with the Wilcoxon test. Comparisons between the parameter data were by the Student t test. All statistical work was done using SPSS (Chicago, Ill). A P-value of less than .05 was taken as statistically significant.

RESULTS

Increased iNOS and Decreased Cleaved Caspase-3 in Lung Cancer of Smokers

The expression of iNOS was significantly increased in the lung tumor tissues of smokers compared with that of nonsmokers, as demonstrated immunohistochemically (Fig. 1A). In contrast to iNOS, the level of cleaved caspase-3 (the active subunit of caspase-3) was lower in the lung tumor tissues of smokers than that of nonsmokers (Fig. 1A). Typical results of the immunohistochemical staining are shown in Figure 1B. The expression of other apoptosis-related molecules, Bcl-2, Bcl-xL, and Bax, was not different between the lung tumor tissues of smokers and nonsmokers (data not shown). The immunohistochemical results were confirmed by Western blot analysis. Figure 1C was 4 typical examples of iNOS protein expression detected by Western blot.

Figure 1. Expression of iNOS and caspase-3 proteins in lung cancer of smokers and nonsmokers are shown. The expression of iNOS and cleaved caspase-3 proteins in lung tumor tissues of smokers and nonsmokers was examined by immunohistochemical staining with their specific antibodies as described in Materials and Methods. Positive staining for iNOS cleaved caspase-3 is in brown. (A) Typical results of the immunohistochemical staining. The expression of iNOS (A-D) and cleaved caspase-3 (E-H) proteins in lung tumor tissues of smokers and nonsmokers is shown. The bottom panel (I,J) was the negative control. (B) The result of immunohistochemical staining was semiquantified according to the standard listed in Table 2. (C) iNOS and caspase-3 proteins were detected by Western blot and 4 representative results are shown. Lanes 1, 3: lung tumor tissues of smokers. Lanes 2, 4: lung tumor tissues of nonsmokers. *P < .05 compared with smokers.

The changes in the protein expression of iNOS and caspase-3 should accordingly be reflected by their activities. In agreement with the high level of iNOS in the lung tumor tissues of smokers, we found a significant increase in NO activity (Fig. 2A). In contrast, the activity of caspase-3 was lower in the lung tumor tissues of smokers than nonsmokers (Fig. 2B), which was consistent with the result of caspase-3 protein expression.

Figure 2. The activities of NO and caspase-3 are depicted. The lysates of lung tumor tissues of smokers and nonsmokers were isolated for measuring (A) NO and (B) caspase-3 activities. The activities of NO and caspase-3 were determined by kits from R&D Systems and Chemicon, respectively. *P < .05 compared with smokers, n = 30 for either smokers or nonsmokers.

NO Decreased the Level of Caspase-3

To obtain a more direct relation between NO and caspase-3, we measured caspase-3 catalytic activity in lung cancer cells treated with 7-hydroxystaurosporine (UCN01), an apoptotic inducer,28 in the presence or absence of SNAP, an NO donor. Before the UCN01 experiment we confirmed that SNAP at 0.1, 0.5, 1, and 1.5 mM could increase the level of NO to 36.7, 45.4, 53.7, and 62.8 mmol/L respectively. Without SNAP the level of NO was 25.2 mmol/L. Treatment of the cells with UCN01 caused a sharp increase in caspase-3 activity that was significantly prevented by pretreatment with SNAP at 0.1 or 0.5 mM (Fig. 3A). However, it was noted that with SNAP concentrations of 1 mM or higher the inhibitory effect disappeared and, furthermore, instead of inhibition, SNAP at high concentrations turned out to stimulate the activity of caspase-3. The result indicates that the high level of NO is harmful to cells; thus, the finding appears to be in line with the double-edge functions of NO in human cells.24, 29

Figure 3. Effect of SNAP, an NO donor, on the level of caspase-3 is illustrated. Lung cancer cells (NCI-H23) were treated with UCN01 to induce caspase-3 in the presence or absence of different concentrations of SNAP. (A) Caspase-3 activity assayed by a kit from R&D Systems. (B) Cleaved caspase-3 protein measured by Western blot. *P < .05, P < .01, compared with cells treated with UCN-01 only. #P < .05, P < .01, compared with control (no treatment).

To examine the effect of NO on the caspase-3 activity more directly we performed an immunoblot analysis for the cleaved caspase-3. The cleaved caspase-3 protein (17 kDa) was readily detected in UCN01-treated cells but its level was markedly reduced in the presence of 0.5 mM SNAP (Fig. 3B). These results suggest that a moderate increase in NO was able to efficiently inhibit the caspase catalytic activity in UCN01-treated lung cancer cells.

To further investigate cell function after alteration of NO we determined cell proliferation after the inhibition of NO by NG-methyl-L-arginine (NMA), an inhibitor of NOS.30 Before the UCN01 experiment we also confirmed that NMA at 2 mM could inhibit NO production from 25.2 mmol/L to 12.8 mmol/L, indicating an inhibitory rate of about 50%. It was found that cell proliferation was not significantly affected by NMA alone (Fig. 4). However, it was significantly reduced when the cells were treated with NMA plus UCN01, especially when UCN-01 was used at 100 nM, a suboptimal concentration.28 These results suggest that the inhibition of NO by NMA itself did not affect cell proliferation but it sensitized the cells to cell death stimulation.

Figure 4. This illustration depicts the effect of NMA, an NO inhibitor, on cell proliferation. The lung cancer cells (NCI-H23) responded to the cell death stimulus UCN01 in terms of their proliferation when the NO was inhibited. The cells were treated with NMA, UCN01, or both in combination for 48 hours. After treatment the cell proliferation was determined by teh MTT cell proliferation assay. **P < .01 compared with cells treated with UCN-01 only.

It is known that the cell death caused by UCN01 is mainly via the promotion of apoptosis.28 A DNA fragmentation assay was used here to test whether the reduced cell proliferation was due to apoptosis. Cells treated with UCN01 showed a significant increase in apoptosis (Fig. 5). SNAP at 0.5 mM reduced the amount of DNA fragments induced by UCN-01, whereas NMA at 2 mM markedly enhanced the amount of UCN-01-induced DNA fragments. These results were in line with that of the caspase-3 activity (Fig. 3). Taken together, our results indicate that an appropriate level of NO can protect cells from apoptosis and the inhibition of NO may exacerbate the apoptosis caused by UCN-01.

Figure 5. Represented are effects of SNAP and NMA on DNA fragmentation. Lung cancer cells (NCI-H23) were treated with 100 nM UCN01 in the presence or absence of different concentrations of 0.5 mM SNAP or 2 mM NMA. DNA fragments were assayed by an ELISA kit from Roche and the assay was performed according to the manufacturer's instructions. *P < .05, **P < .01, compared with control (no treatment). #P < .05, ##P < .01, compared with cells treated with UCN-01 only.

NNK Increased the Expression of iNOS

To have experimental data to support the causative relation between cigarette smoking and iNOS in lung cancer, we analyzed the effect of NNK, 1 of the most potent tobacco-specific carcinogens,3 on the proliferation of lung cancer cells and the expression of iNOS in the cell. The result showed that NNK stimulated the proliferation of the cells in a dose- and time-dependent manner (Fig. 6A,B). Using Western blot, we showed that the level of iNOS protein was increased by NNK in a time-dependent fashion (Fig. 6C), with a maximal effect at 24 to 48 hours after NNK treatment. Because NO is the major product of iNOS, it is commonly accepted that an increase in iNOS reflects the elevated level of NO generation.

Figure 6. Effect of NNK on cell proliferation and the expression of iNOS is illustrated. (A) Lung cancer cells (NCI-H23) were treated with various concentrations of NNK for 48 hours and the cell proliferation was determined by the MTT cell proliferation assay. (B) The cells were treated with 10 mM NNK for 24, 48, and 72 hours before the cell proliferation was performed. (C) The cells were treated with 10 mM NNK for different periods of time. After the treatment, proteins were isolated and subjected to Western blot for iNOS protein detection. *P < .05, **P < .01, compared with cells without NNK treatment.

DISCUSSION

One of the most significant findings in the present study is the increased iNOS in lung cancer tissues from patients who smoke, compared with that from patents who do not smoke. We have also shown a significant increase in NO, the enzymatic product of iNOS, as measured by its stable metabolites, nitrite and nitrate, in lung cancer tissues from patients who smoke relative to the tissues from those who do not smoke. We have previously shown an increase in iNOS in human lung cancer,25 but failed to identify a significant relation with cigarette smoking, which is most likely due to the small number of patients tested, especially the patients who smoke. Several other groups have also reported an elevated level of iNOS or NO in lung cancer8–10, 31 but the relation with cigarette smoking has not been studied. To the best of our knowledge, our study is the first report to reveal a relation between cigarette smoking and increased iNOS/NO in lung cancer tissues.

Cigarette smoking can affect cellular metabolism in various types of cells and tissues in which the physiologic level of NO functions to maintain cell proliferation and growth. To investigate how cigarette smoking contributes to the pathologic process, a number of studies have been performed to examine the effect of cigarette smoking on NO. In gastric mucosa, cigarette smoking stimulates NO production.32 Tobacco extract induces the production of NO in hamster cheek pouch cells.16 However, cigarette smoking has also been noted to reduce the level of NO in other types of cells such as blood monocytes and platelets.19, 20 There is also evidence that cigarette smoking does not affect iNOS expression in airway epithelial cells of normal subjects who smoke.21 Therefore, it appears that the effect of cigarette smoking on iNOS/NO varies depending on the types of cells tested and the designs of the experiments. In our study the lung cancer tissues studied were from patients who at least had a 10-year experience of smoking and the average smoking history was 38 years. The increase of iNOS protein was demonstrated by both immunochemical staining and Western blot analysis. The increased iNOS protein was further supported by the elevated NO activity detected in the same set of samples. In addition to the lung cancer tissue study, we used a NSCLC cell line (NCI-H23) to test the relation between cigarette smoking and iNOS expression. The level of iNOS protein was obviously increased in the cells treated with NNK, the most potent tobacco-specific carcinogen. The increase of iNOS protein was accompanied by elevated cell proliferation. NNK is able to induce noncancerous human epithelial cells to adopt malignant features and the transformed cells are tumorigenic in immunodeficient mice.24 Furthermore, NNK can consistently induce lung cancer in various animal models.3 There are also experiments demonstrating that NO contributes to the development of cancer.33 Therefore, NNK may reduce caspase-3 activity and thus apoptosis by stimulating iNOS, which contributes to the proliferation and growth of lung cancer cells.

The role of NO in carcinogenesis appears to be complex due to its divergent functional activities. One of the important roles for NO is its involvement in the regulation of cell growth and apoptosis. NO inhibits the activation of apoptosis signal-regulating kinase 1 to reduce apoptosis in murine fibrosarcoma cells.30 NO suppression results in apoptosis via a pathway related to the activation of FKHRL1 and ROCK kinases in human breast cancer cells,34 suggesting an antiapoptotic role of NO. NO can also protect osteoblast cells from apoptosis via enhancing the level of heme oxygenase-1.35 In human colon cancer cells, NO suppresses apoptosis by scavenging mitochondrial superoxide anions and by down-regulating antiapoptotic Bcl-xL.36 In our study we demonstrated that the level of the cleaved caspase-3 and the amount of DNA fragments were significantly reduced in lung cancer cells treated by the NO donor SNAP, indicating an inhibitory role of NO in the activity of caspase-3 and in the generation of DNA fragmentation. The finding is in agreement with the discrepant expression pattern of the increased iNOS and the decreased cleaved caspase-3 in lung cancer tissues of smokers found in this study. The result may suggest that lung cancer cells potentially grow without apoptotic control due to an increase in NO and the low level of caspase-3. Our study further demonstrated that the apoptosis induced by apoptotic stimulus UCN-01 was further enhanced when the generation of NO was blocked by its inhibitor NMA, as evident by the increased activity of caspase-3 as well as the elevated amount of DNA fragments. Interestingly, the lung cancer cells with a low level of NO, due to NMA treatment alone, did not show a significant increase in apoptosis. However, apoptosis in these cells was markedly induced by a cell death inducer, UCN01, suggesting that the inhibition of NO would greatly increase the sensitivity of lung cancer cells to death stimulation. The finding also provides a rationale to use NO inhibitors to enhance the efficiency of chemotherapy in lung cancer. The finding that the inhibition of NO production by NMA alone fails to increase apoptosis may suggest that lung cancer cells have a compensatory mechanism to cover the loss of NO induced by NMA; however, initiation of such a compensatory reserve may reduce the ability of cells to resist potential death stimulation.

In contrast to the inhibition of apoptosis by NO, numerous reports also indicate that NO can kill tumor cells via the promotion of apoptosis.29, 37 This apparent paradox, in which NO both enhances and inhibits tumorigenesis, has been attributed to a number of factors including local NO concentrations, cell types, cellular genetics, and redox status within a cell. At low levels, NO appears to increase the tumor-promoting effects, whereas at high levels it is cytotoxic. Our study provides some evidence to support NO as a bioregulator of apoptosis. We found that the caspase-3 activity and DNA fragmentation induced by UCN01 were significantly inhibited by pretreatment of the cells with the NO donor SNAP. However, such an inhibitory effect disappeared when SNAP was used at concentrations of 1 mM or higher. The high concentration of SNAP may even stimulate instead of suppress the activity of caspase-3. It is reported that SNAP at concentrations between 0.1 and 1 mM may release free NO in the range of 20-100 nM, which is thought to be physiologically relevant concentrations.38 Therefore, the net effect of NO on apoptosis or cell growth in a given cell type is complex and finely tuned.

In conclusion, cigarette smoking stimulates the production of NO, probably via the up-regulation of iNOS, in lung cancer cells. Carcinogenic chemical compounds of cigarettes, at least NNK, contribute to the promotion of NO in lung cancer. The increased iNOS/NO may enable the lung cancer cells to escape apoptosis via the down-regulation of caspase-3, and thus enhance the ability of lung cancer cells to proliferate and grow.