WTA 2014 PLENARY PAPER
Estrogen modulates intestinal mucus physiochemical properties and protects against oxidant injury Mark E. Diebel, MD, Lawrence N. Diebel, MD, Charles W. Manke, PhD, and David M. Liberati, MS, Detroit, Michigan
BACKGROUND: The intestinal epithelial barrier and the intestinal mucus layer may be protective against trauma/hemorrhage shockYinduced injury in females. This effect is related to estradiol (E2) concentrations and varies with the menstrual cycle. We examined the ability of E2 to impact the physiochemical properties of intestinal mucus and to protect against oxidant-related injury to the mucus and underlying intestinal epithelial barrier in an in vitro model. METHODS: NonYmucus-producing (HT29) and mucus-producing (HT29-MTX) intestinal epithelial cells (IECs) were exposed to E2 or no E2 for 3 days and then grown to confluence on transwell plates. Nonadherent and adherent mucus content was indexed by analysis of mucin using an enzyme-linked immunosorbent assay and mucus viscosity (cp) and elasticity (G’) were determined by rheometry. In additional experiments, IEC groups were exposed to hydrogen peroxide and IEC apoptosis as well as permeability (fluorescein isothiocyanateYdextran) and oxidative damage determined by measuring lipid hydroperoxide and protein carbonyl content. RESULTS: There were nearly 50% increases in the mucin content of both the nonadherent and adherent mucus layer(s) in HT29-MTX cells exposed to estrogen. Estrogen treatment also resulted in a twofold and eightfold increase in mucus viscosity and elasticity versus HT29-MTX cells with no estrogen exposure, respectively. Oxygen radical damage to the mucus layer caused by H2O2 was significantly reduced by E2 compared with HT29-MTX + H2O2 without estrogen. Estrogen treatment resulted in significant reductions in both apoptosis and permeability seen after H2O2 challenge. CONCLUSION: The results of this study suggest that sex differences in gut barrier function following trauma/hemorrhage shock may in part be related to differences in intestinal mucus content and the resultant physiochemical and oxidant-resistant properties of the mucus layer. (J Trauma Acute Care Surg. 2015;78: 94Y99. Copyright * 2015 Wolters Kluwer Health, Inc. All rights reserved.) KEY WORDS: Estrogen; mucin; HT29-MTX cells; oxidant injury.
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here is experimental evidence that estrogen is responsible for the protection against gut injury following trauma/ hemorrhagic shock (T/HS).1,2 Caruso et al.3 have shown that the effect was most pronounced in rats during the proestrus phase of the estrus cycle, where estrogen levels are the highest. Subsequent studies by their laboratory demonstrated that the resistance of female rats to T/HS-induced intestinal and lung injury was associated with better preservation of the intestinal mucus layer.4 However, the mechanisms remain speculative. The anatomic and physiologic properties of gastrointestinal mucus are a subject of increasing research interest.5Y9 This is important because it is recognized that the mucus layer is the first line of innate host defense at gastrointestinal mucosal surfaces largely because of mucin glycoprotein’s secreted by intestinal goblet cells.10,11 In this regard, disruption in the intestinal mucus layer following T/HS has been anatomically and causally linked to injury to the underlying intestinal epithelial cell (IEC) barrier.
Gut ischemia-reperfusion insults are associated with oxygen radical production. Oxyradical scavenging by gastrointestinal mucus has been demonstrated in in vitro studies, which may have a protective effect on the underlying mucosal epithelial cells.12Y14 However, other studies have shown oxidant-related injury to the intestinal mucus layer following T/HS, which correlated with injury to the associated gut mucosal barrier.15,16 The protective effect of the intestinal mucus layer depends on the thickness and stability as well as the viscoelastic properties of the mucus gel.5,6 Mucus viscoelasticity is largely influenced by mucin concentration as gel viscosity varies exponentially with mucin concentration at a constant shear rate.6 Estrogen has been shown to increase mucoprotein content of cervical mucus and increase viscosity-related barrier protection.6 We therefore studied the effect of estrogen on intestinal mucus viscoelastic properties and protection against oxidant injury to the mucus and underlying intestinal mucosal layer in an in vitro model.
Submitted: January 28, 2014, Revised: August 27, 2014, Accepted: October 1, 2014. From the Departments of Surgery (M.E.D., L.N.D., D.M.L.) and Chemical Engineering (C.W.M.), Wayne State University, Detroit, Michigan. This study was presented at the Western Trauma Association meeting, March 2Y7, 2014, in Steamboat Springs, Colorado. Address for reprints: Lawrence N. Diebel, MD, Department of Surgery, 6C-University Health Center, 4201 Saint Antoine, Detroit, MI 48201; email: [email protected].
MATERIALS AND METHODS
DOI: 10.1097/TA.0000000000000499
IECs (NonYMucus-Producing Clone) HT29 cells, isolated from a colorectal adenocarcinoma from an adult female, were obtained from American Type Culture Collection and routinely cultured with Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum, 4.5-g/L glucose, and gentamicin in an atmosphere of 5% CO2 J Trauma Acute Care Surg Volume 78, Number 1
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at 37-C. Cells (5 105) were seeded on the apical surface of a polycarbonate membrane (3.0-Km pore size) (Transwell, Corning Costar Core, Cambridge, MA) in a two-chamber cell culture system and allowed to form polarized monolayers. Monolayer integrity was monitored by serial measurement of the transepithelial electrical resistance with a Millicell electrical resistance meter (Millipore Corp., Bedford, MA). When grown in this system, HT29 cells form polarized monolayers that are confluent after 10 days to 14 days in culture.
HT29-MTX Cells (Mucus-Producing Clone) The HT29-MTX-E12 cell line was obtained from HPA Cultures (Salisbury, United Kingdom) and routinely cultured with Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum, 4.5-g/L glucose, 1% nonessential amino acids, and 1% antibiotic/antimycotic (Gibco, Grand Island, NY) in an atmosphere of 5% CO2 at 37-C. Cells (5 105/mL) were seeded on the apical surface of a polycarbonate membrane (3.0-Km pore size) as described for HT29 cells earlier. This subpopulation of HT29 cells were differentiated into columnar absorptive cells and mucus secreting cells.17
Experimental Design HT29 and HT29-MTX IECs were grown to confluence in tissue culture flasks in the presence or absence of 90-pg/mL estrogen for up to 3 days. The cells were subsequently trypsinized and seeded onto two-chamber cell culture plates and grown to confluence. The nonadherent and adherent mucus layers were isolated, and mucus content was quantified by enzyme-linked immunosorbent assay (ELISA) specific for mucin 1 (MUC 1). In other experiments, the effect of estrogen on the viscosity and elasticity of the mucus layer was analyzed using rheologic testing. In a separate set of experiments, exposure of the IEC to 250-KM hydrogen peroxide (H2O2) for 30 minutes was introduced to assess the ability of estrogen to protect the intestinal barrier against oxidant-related injury. IEC apoptosis and permeability after H2O2 exposure were quantified by flow cytometry. Oxidative damage to IEC monolayers was measured by lipid hydroperoxide release and protein carbonyl content.
O-Linked Oligosaccharide Quantification Nonadherent mucin quantification was determined using a modification of the assay developed by Tanabe et al.18 Briefly, HT29 and HT-29 MTX cell monolayers and those treated with 90-pg/mL estrogen for 3 days were washed with phosphatebuffered saline (PBS) several times. These washings were then centrifuged at 10,000 G for 30 minutes, and the supernatants were removed. Ice cold ethanol at 60% by volume was added, and the samples were allowed to precipitate. The mucin fraction was recovered by centrifugation at 1,400 G for 10 minutes and finally dissolved in 5-mL dH20. The nonadherent mucin content was finally measured via fluorescence at 383 nm. Standard solutions of N-acetylgalactosamine (Sigma, St. Louis, MO) were used to calculate the amount of O-linked oligosaccharide chains liberated from mucins during the procedure.
Alcian Blue Quantification of Mucins Determination of the total amount of adherent mucin on the surface of HT29-MTX cells was quantitated using methods
similar to those outlined by Scott and Dorling19 and Scott and Willett.20 Briefly, HT-29 MTX cells with or without estrogen and washed free of nonadherent mucin with PBS were mixed with 1 mL of Alcian blue reagent (0.5% alcian blue in 50-mM sodium acetate containing 50-mM MgCl2). This mixture incubates for 2 hours at room temperature, after which the alcian blueYmucus complex was centrifuged at 2,000 G for 15 minutes. Once the precipitate is dissolved, the adherent mucus content was determined using an ELISA specific for MUC 1. Standards were prepared using chondroitin sulfate over the range of 1 Kmol to 10 Kmol.
MUC 1 ELISA MUC 1 content was quantitated in both the nonadherent and adherent mucin collected from HT-29 MTX cells using an ELISA assay kit specific for MUC 1 purchased from USCNK Life Science, Inc. (Wutnan, China). Standards and samples are run according to the kit instructions, and the amount of MUC 1 present in the samples is determined by comparing the optical density of the samples to the standard curve measured at 450 nm.
Rheology Testing To evaluate the viscoelastic properties of HT29 and HT29-MTX cells exposed to 90 pg/mL estrogen (E2) for 3 days, the IECs were grown on glass cover slips (2.5-cm circles) in 6-well plates (Fisher Scientific, Hanover Park, IL) at a concentration of 1 105 cells/mL. Once cells reach confluence on the cover slips, they are mounted with super glue (cyanoacrylate adhesive) directly to the lower platen of a Rheometrics ARES rheometer equipped with 2.5-cm parallel plates. The cell culture samples are then tested in small amplitude oscillatory shear flow to measure the storage modulus G’ (U) and loss modulus G’’ (U), which characterize the linear viscoelastic response of the fluid layer. All rheologic testing is conducted at a temperature of 37-C, controlled by a humidified environmental chamber, and at 0.5% strain, which assures that the mucus layer response remains in the linear viscoelastic regimen. Each sample is tested at oscillatory frequencies ranging from 0.1 rad/s to 100 rad/s (0.016Y16 Hz). Variation of viscoelasticity with position within the mucus layer causes a nonuniform strain within the mucus layer. Therefore, the measured G’ and G’’ values must be interpreted as apparent, rather than absolute, values of these viscoelastic material functions. Viscosity measurements of mucus samples washed from the surface of HT29 and HT29-MTX cell cultures were conducted with a Contraves LS-30 rheometer at 13 per second, using cup-and-bob fixtures.
Determination of Lipid Peroxide Malondialdehyde (MDA), a byproduct of lipid peroxide, was detected in HT29-MTX cells using an MDA Adduct ELISA Kit (Cell Biolabs, Inc., San Diego, CA). The kit was used according to manufacturer’s instruction and has a sensitivity limit of 2-pmol/mg MDA Adduct.
Determination of Protein Carbonyl Content Protein carbonyl content in HT29-MTX cells was calculated using a Protein Carbonyl Spectrophotometric Assay Kit (Cell Biolabs, Inc.). Cell samples were run according to the
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manufacturer’s instructions, and the protein carbonyl content was calculated. Data are expressed as nanomole per milligram.
Measurement of Reactive Nitrogen Intermediates Nitration of tyrosine residues, a marker of reactive nitrogen intermediate (RNI)Ymediated injury, was measured using the OxiSelect Nitrotyrosine ELISA kit (Cell Biolabs Inc.). Mucus samples were added according to the kit instructions, and nitrated bovine serum albumin (BSA) standards were also plated to provide a known nitrated BSA standard curve. The protein nitrotyrosine content in the samples is determined by comparison with the nitrated BSA standards provided.
Apoptosis Confluent HT29-MTX cells from the different treatment groups were analyzed for apoptosis using the TACS Annexin-V Apoptosis Detection Kit (R&D Systems, Minneapolis, MN). HT29-MTX cells were incubated with an Annexin-V Incubation reagent for 15 minutes in the dark then mixed with a 1 binding buffer and analyzed by flow cytometry within 1 hour for maximal signal. Results were expressed as percent apoptosis compared with control cells.
Permeability Determination Using Fluorescein IsothiocyanateYDextran Confluent HT29-MTX cells with or without estrogen grown on inserts were exposed to 250-KM H2O2 for up to 30 minutes. Fluorescein isothiocyanate (FITC)Ydextran (4,000 molecular weight, 2.2 mg/mL in PBS) was added to each insert, and the monolayer was incubated for 2 hours at 37-C. Monolayer permeability to FITC-dextran was then determined by measuring the fluorescence of the basal solution in each well at 485 nm (excitation wavelength) and 535 nm (emission wavelength). Permeability data were expressed as nanomole per square centimeter per hour.
Statistical Analysis An analysis of variance with a post hoc Tukey test was used to analyze the data. Statistical significance was inferred at p values of less than 0.001. All data are expressed as mean (SD).
RESULTS The effect of treatment of HT29-MTX cells with estrogen led to significant increases in the mucin content of the mucus layer (Fig. 1A and B). There were 45% to 47% increases in the mucin content in the nonadherent and adherent mucus layers, respectively (p G 0.001 vs. no estrogen HT29-MTX group). The impact of estrogen treatment on the viscoelastic properties of the mucus layer are shown in Figure 2A and B. Estrogen treatment resulted in a twofold increase in viscosity and a nearly eightfold increase in elasticity of the mucus layer (p G 0.001 vs. no estrogen HT29-MTX group). The data shown in Figure 2A and B reflect a shear stress typical of the intestine in a fasting state. The protective effect of estrogen on oxygen radical damage to the mucus layer is demonstrated in Figure 3. Both protein oxidation (protein carbonyl) and lipid oxidation (lipid hydroperoxide) products were determined. Exposure to H2O2 led to a five times increase in lipid hydroperoxide and a three times increase in protein carbonyl content (Fig. 3A, p G 0.001 vs. 96
Figure 1. (A), The effect of estrogen pretreatment on nonadherent mucin content in HT29-MTX cells. The nonadherent mucus layer was isolated from HT29-MTX cells with or without 90-pg/mL estrogen for up to 3 days. The nonadherent component of the mucus layer was measured using fluorescence at 383 nm. Known N-acetylgalactosamine standards were used to quantify O-linked oligosaccharide chains liberated from the mucin layer. (B), The effect of estrogen pretreatment on adherent mucus content of HT29-MTX cells. The adherent mucus layer was isolated from HT29-MTX cells with or without 90-pg/mL estrogen for up to 3 days. The protein MUC 1 was quantified by ELISA with an antibody specific for the MUC 1 protein.
HT29-MTX without H2O2 treatment). Pretreatment with estrogen resulted in the significant reduction of both of these oxidant-related markers to near-baseline levels (p G 0.001 vs. HT29-MTX + H2O2). RNIs were increased nearly two times in HT29-MTX cells treated with H2O2 (Fig. 3B, p G 0.001). However, these levels were reduced to RNI concentrations of nonYH2O2-treated HT29-MTX cells. The effect of luminal H2O2 challenge on the underlying HT29 monolayer was indexed by apoptosis and permeability (Figs. 4 and 5). There was a greater than three times increase in HT29-MTX apoptosis and a twofold increase in monolayer permeability after exposure to H2O2. Estrogen pretreatment resulted in marked reductions in apoptosis and permeability noted after H2O2 challenge (p G 0.001 vs. HT29-MTX + H2O2).
DISCUSSION There is increasing recognition of the role of the mucus layer covering the gastrointestinal epithelium in protection * 2015 Wolters Kluwer Health, Inc. All rights reserved.
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of the mucus layer after H2O2 challenge. In this regard, Brownlee et al.14 failed to demonstrate any changes in the absolute value of storage and loss moduli of colonic mucus after reactive oxygen species (ROS) treatment (50-nM H2O2). However, because mucus is not a homogeneous fluid, the macroscopic viscoelastic measurements are an average assessment of the rheology of nanoscopically heterogeneous environments of the mucus layer. Thus, recent studies have focused on the importance of mucus microrheology to better understand the biophysical properties of mucus.22 Previous studies have shown that estrogen protects against T/HS intestinal injury and remote organ failure.3 This may in part be related to a cytoprotective effect by activation of functional estrogen receptors on IEC.23 In another study, Sheth et al. demonstrated that there was preservation of the intestinal mucus layer in females and postulated that this may have a
Figure 2. (A), The effect of estrogen pretreatment on mucus layer viscosity in HT29-MTX cells. HT29 and HT29-MTX cell monolayers and those treated with 90-pg/mL estrogen for 3 days were washed with PBS several times to remove the mucus layer. The washings were subjected to rheologic testing using a Contraves LS-30 rheometer. Viscosity is expressed as centipoises (Cp) at a shear rate of 13 per second. (B), Analysis of the elasticity of the mucus layer in HT29-MTX cells pretreated with estrogen. HT29 and HT29-MTX cells exposed to 90-pg/mL estrogen (E2) for 3 days were grown on glass cover slips and mucus storage modulus (G’) measured using the ARES rheometer. G’ values, representing the elastic response of the mucus layer, were measured at a frequency of 10 rad/s. and 1% strain at a position 75 Km above the substrate surface.
against gastric acid, pancreatic digestive enzymes, and luminal bacteria especially under stress conditions.15,16,21 Mucins are highly glycosylated proteins and are the major organic component of mucus.9,10 The viscoelastic properties of mucus are primarily due to mucin/water content and secondarily to lipids, other proteins, and iron content.6 The importance of mucin concentration on mucus viscoelasticity is illustrated by cervical mucus.6 The mucin concentration in nonovulatory mucus has been noted to increase by only twofold or fourfold; however, this markedly increased its viscoelasticity, making it virtually impenetrable to sperm. In our study, exposure to physiologic concentrations of estrogen increased mucin content of the mucus layer and led to marked changes in the rheologic properties of the mucus layer. In our study, the elastic component of the viscoelastic shear modulus (G’) and the viscous component (G’’) were increased by estrogen treatment. As a non-Newtonian fluid, this varied with the shear stress but was apparent over a wide range of the shear stress applied. We did not obtain rheologic measurements
Figure 3. (A), The effect of H2O2 on oxidative damage to the mucus layer of IEC monolayers: measurement of lipid hydroperoxide and protein carbonyl content. A by-product of lipid peroxide, MDA, was measured in the mucus layer collected from HT29-MTX cells using an MDA Adduct ELISA Kit. Protein carbonyl content was measured in the mucus layer of HT29-MTX cells exposed to 90-pg/mL estrogen for 3 days and 250-KM H2O2 for up to 30 minutes using a Protein Carbonyl Spectrophotometric Assay Kit. (B), The effect of H2O2 on oxidative damage to the mucus layer of IEC monolayers: measurement of RNIs. Oxidative damage mediated by RNI increased in the mucus layer isolated from HT29-MTX cells exposed to 250-KM H2O2 for 30 minutes. Pretreatment of HT29-MTX cells with 90-pg/mL estrogen for 3 days reduced the damage from RNI in the mucus layer.
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Figure 4. The protective effect of estrogen on apoptosis after H2O2 treatment of HT29-MTX cells. HT29-MTX cells were cocultured with 90-pg/mL estrogen for 3 days followed by exposure to 250-KM H2O2 for 30 minutes. Apoptosis was quantified using flow cytometry.
protective effect against shock-induced gut injury and subsequent remote organ injury. In our study, estrogen treatment of HT29 cells led to changes in the rheologic properties of the mucus layer and increased mucus content. Oxidant injury of the intestinal mucus layer may be important in the pathogenesis of T/HS gut barrier failure.24 Because of the protective effects of estrogen on T/HS gut barrier failure and the physiochemical changes in the mucus layer noted in our study, we studied the effect of a standard oxidant challenge on the mucus layer and intestinal barrier function. Our studies demonstrated significantly decreased protein carbonyl, nitrated tyrosine residues, and lipid peroxidation in HT29-MTX cells treated with estrogen and then exposed to H2O2. This may reflect enhanced antioxidant properties of the mucus layer because of increased mucin content. Other possibilities include other genomic and nongenomic effects of estrogen on IECs and/or mucus secreting cells. Native mucus gel has been shown to be more resistant to ROS damage than purified mucin.14 Thus, nonmucin components of the mucus may also have ROS-scavenging properties.
Figure 5. Estrogens effect on IEC monolayer permeability after H2O2 treatment. Monolayer permeability to FITC-dextran (FD4) was determined after HT29-MTX cells were exposed to 90-pg/mL estrogen for 3 days and 250-KM H2O2 for up to 30 minutes. 98
Although lipids such as phosphatidylcholine make up only a small percentage of intestinal mucus, they may be important in the protection against oxygen radical damage to mucin. Of note, most of the lipids in mucus is associated with hydrophobic domains of the mucin glycoproteins.6 Exogenous H2O2 was used to provide a uniform oxidant challenge to the mucus layer and the IECs in our model. Endogenous sources of oxyradicals following gut perfusion injury in vitro include IEC xanthine oxidase and phospholipase A2.25,26 Of note, the action of phospholipase A2 on the lipid component of the mucus layer (phosphatidylcholine) may increase oxygen radical damage to mucins as lipid binding to mucin has a protective effect against oxygen radicals.13 Additional studies on the effect of estrogen on the lipid component of the mucus layer before and after oxygen challenge are therefore warranted. In summary, estrogen treatment increased mucin content and enhanced the rheologic properties of the mucus layer of HT29-MTX cells. This afforded protection to the mucus layer and the underlying intestinal epithelial monolayer following oxidant challenge. The protective effect of estrogen against oxidative stress likely includes genomic and nongenomic pathways.1 Our study suggests that this in part is due to enhanced rheologic and oxygen scavenging properties of the mucus layer following treatment with estrogen. AUTHORSHIP L.N.D., D.M.L., M.E.D., and C.W.M. designed this study. L.N.D., D.M.L., M.E.D., and C.W.M. performed the data collection, data analysis, statistical analysis, and data interpretation. L.N.D., D.M.L., M.E.D., and C.W.M. prepared the figures. L.N.D., D.M.L., M.E.D., and C.W.M. wrote and critically revised the article. L.N.D., D.M.L., M.E.D., and C.W.M. claim overall responsibility.
DISCLOSURE The authors declare no conflicts of interest.
REFERENCES 1. Kawasaki T, Chaudry IH. The effects of estrogen on various organs: therapeutic approach for sepsis, trauma, and reperfusion injury. Part 2: liver, intestine, spleen, and kidney. J Anesth. 2012;26:892Y899. 2. Haider AH, Crompton JG, Chang DC, Efron DT, Haut ER, Handly N, Cornwell EE. Evidence of hormonal basis for improved survival among females with trauma-associated shock: an analysis of the National Trauma Data Bank. J Trauma. 2010;69(3):537Y540. 3. Caruso JM, Deitch EA, Xu DZ, Lu Q, Dayal SD. Gut injury and gutinduced lung injury after trauma hemorrhagic shock is gender and estrus cycle specific in the rat. J Trauma. 2003;55(3):531Y539. 4. Sheth SU, Lu Q, Twelker K, Sharpe SM, Qin X, Reino DC, Lee MA, Xu DZ, Deitch EA. Intestinal mucus layer preservation in female rats attenuates gut injury after trauma-hemorrhagic shock. J Trauma. 2010;68(2): 279Y288. 5. Atuma C, Strugala V, Allen A, Holm L. The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo. Am J Physiol Gastrointest Liver Physiol. 2001;280:G922YG929. 6. Cone RA. Barrier properties of mucus. Adv Drug Deliv Rev. 2009;61: 75Y85. 7. Ermund A, Schu¨tte A, Johansson ME, Gustafsson JK, Hansson GC. Studies of mucus in mouse stomach, small intestine, colon. I. Gastrointestinal mucus layers have different properties depending on location as well as over the Peyer’s patches. Am J Physiol Gastrointest Liver Physiol. 2013;305:G341YG347.
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8. Johansson MEV, Ambort D, Pelaseyed T, Schu¨tte A, Gustafsson JK, Ermund A, Subramani DB, Holme´r-Larsson JM, Thomsson KA, Bergstro¨m JH, et al. Composition and functional role of the mucus layers in the intestine. Cell Mol Life Sci. 2011;68:3635Y3641. 9. Dharmani P, Srivastava V, Kissoon-Singh V, Chadee K. Role of intestinal mucins in innate host defense mechanisms against pathogens. J Innate Immun. 2009;1:123Y135. 10. Kims YS, Ho SB. Intestinal goblet cells and mucins in health and disease: recent insights and progress. Curr Gastroenterol Rep. 2010;12(5):319Y330. 11. Johansson MEV, Hansson GC. The goblet cell: a key player in ischaemiareperfusion injury. Gut. 2013;62(2):188Y189. 12. Grisham MB, Von Ritter C, Smith BF, Lamont JT, Granger DN. Interaction between oxygen radicals and gastric mucin. Am J Physiol. 1987;253: G93YG96. 13. Gong D, Turner B, Bhaskar KR, Lamont JT. Lipid binding to gastric mucin: protective effect against oxygen radicals. Am J Physiol. 1990;259: G681YG686. 14. Brownlee IA, Knight J, Dettmar PW, Pearson JP. Action of reactive oxygen species on colonic mucus secretions. Free Radic Biol Med. 2007;43: 800Y808. 15. Qin Z, Caputo FJ, Zu DZ, Deitch EA. Hydrophobicity of mucosal surface and its relationship to gut barrier function. Shock. 2008;29(3):372Y376. 16. Rupani B, Caputo FJ, Watkins AC, Vega D, Magnotti LJ, Lu Q, Xu da Z, Deitch EA. Relationship between disruption of the unstirred mucus layer and intestinal restitution in loss of gut barrier function after trauma hemorrhagic shock. Surgery. 2007;141:481Y489. 17. Lesuffleur T, Barbat A, Dussaulx E, et al. Growth adaptation to methotrexate of HT29 human colon carcinoma cells is associated with their
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ability to differentiate into columnar absorptive and mucus-secreting cells. Cancer Res. 1990;50:6334Y6343. Tanabe H, Ito H, Sugiyama K. Estimation of luminal mucin content in rats by measurement of O-linked oligosaccharide chains and direct ELISA. Biosci Biotechnol Biochem. 2007;71(2):575Y578. Scott JE, Dorling J. Differential staining of acid glycosaminoglycans by alcian blue in salt solutions. Histochemie. 1965;5(3):221Y233. Scott JE, Willett H. Binding of cationic dyes to nucleic acids and their biological polyanions. Nature. 1966;209:985Y987. Qin Z, Sheth SU, Sharpe SM, Dong W, Lu Q, Xu D, Deitch EA. The mucus layer is critical in protecting against ischemia/reperfusion-mediated gut injury and in the restitution of gut barrier function. Shock. 2011;35(3): 275Y281. Lai SK, Wang YY, Wirtz D, Hanes J. Micro- and macrorheology of mucus. Adv Drug Deliv Rev. 2009;61(2):86Y100. Thomas ML, Xu X, Norfleet AM, Watson CS. The presence of functional estrogen receptors in intestinal epithelial cells. Endocrinology. 1993;132(1): 426Y430. Fishman JE, Levy G, Alli V, Sheth S, Lu Q, Deitch EA. Oxidative modification of the intestinal mucus layer is a critical but unrecognized component of trauma hemorrhagic shock-induced gut barrier failure. Am J Physiol Gastrointest Liver Physiol. 2013;304:G57YG63. Granger DN. Role of xanthine oxidase and granulocytes in ischemiareperfusion injury. Am J Physiol. 1988;255:H1269YH1275. Otamiri T, Franze´n L, Lindmark D, Tagesson C. Increased phospholipase A2 and decreased lysophospholipase activity in the small intestinal mucosa after ischaemia and revascularization. Gut. 1987;28:1445Y1453.
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Estrogen modulates intestinal mucus physiochemical properties and protects against oxidant injury Mark E. Diebel, MD, Lawrence N. Diebel, MD, Charles W. Manke, PhD, and David M. Liberati, MS, Detroit, Michigan
BACKGROUND: The intestinal epithelial barrier and the intestinal mucus layer may be protective against trauma/hemorrhage shockYinduced injury in females. This effect is related to estradiol (E2) concentrations and varies with the menstrual cycle. We examined the ability of E2 to impact the physiochemical properties of intestinal mucus and to protect against oxidant-related injury to the mucus and underlying intestinal epithelial barrier in an in vitro model. METHODS: NonYmucus-producing (HT29) and mucus-producing (HT29-MTX) intestinal epithelial cells (IECs) were exposed to E2 or no E2 for 3 days and then grown to confluence on transwell plates. Nonadherent and adherent mucus content was indexed by analysis of mucin using an enzyme-linked immunosorbent assay and mucus viscosity (cp) and elasticity (G’) were determined by rheometry. In additional experiments, IEC groups were exposed to hydrogen peroxide and IEC apoptosis as well as permeability (fluorescein isothiocyanateYdextran) and oxidative damage determined by measuring lipid hydroperoxide and protein carbonyl content. RESULTS: There were nearly 50% increases in the mucin content of both the nonadherent and adherent mucus layer(s) in HT29-MTX cells exposed to estrogen. Estrogen treatment also resulted in a twofold and eightfold increase in mucus viscosity and elasticity versus HT29-MTX cells with no estrogen exposure, respectively. Oxygen radical damage to the mucus layer caused by H2O2 was significantly reduced by E2 compared with HT29-MTX + H2O2 without estrogen. Estrogen treatment resulted in significant reductions in both apoptosis and permeability seen after H2O2 challenge. CONCLUSION: The results of this study suggest that sex differences in gut barrier function following trauma/hemorrhage shock may in part be related to differences in intestinal mucus content and the resultant physiochemical and oxidant-resistant properties of the mucus layer. (J Trauma Acute Care Surg. 2015;78: 94Y99. Copyright * 2015 Wolters Kluwer Health, Inc. All rights reserved.) KEY WORDS: Estrogen; mucin; HT29-MTX cells; oxidant injury.
T
here is experimental evidence that estrogen is responsible for the protection against gut injury following trauma/ hemorrhagic shock (T/HS).1,2 Caruso et al.3 have shown that the effect was most pronounced in rats during the proestrus phase of the estrus cycle, where estrogen levels are the highest. Subsequent studies by their laboratory demonstrated that the resistance of female rats to T/HS-induced intestinal and lung injury was associated with better preservation of the intestinal mucus layer.4 However, the mechanisms remain speculative. The anatomic and physiologic properties of gastrointestinal mucus are a subject of increasing research interest.5Y9 This is important because it is recognized that the mucus layer is the first line of innate host defense at gastrointestinal mucosal surfaces largely because of mucin glycoprotein’s secreted by intestinal goblet cells.10,11 In this regard, disruption in the intestinal mucus layer following T/HS has been anatomically and causally linked to injury to the underlying intestinal epithelial cell (IEC) barrier.
Gut ischemia-reperfusion insults are associated with oxygen radical production. Oxyradical scavenging by gastrointestinal mucus has been demonstrated in in vitro studies, which may have a protective effect on the underlying mucosal epithelial cells.12Y14 However, other studies have shown oxidant-related injury to the intestinal mucus layer following T/HS, which correlated with injury to the associated gut mucosal barrier.15,16 The protective effect of the intestinal mucus layer depends on the thickness and stability as well as the viscoelastic properties of the mucus gel.5,6 Mucus viscoelasticity is largely influenced by mucin concentration as gel viscosity varies exponentially with mucin concentration at a constant shear rate.6 Estrogen has been shown to increase mucoprotein content of cervical mucus and increase viscosity-related barrier protection.6 We therefore studied the effect of estrogen on intestinal mucus viscoelastic properties and protection against oxidant injury to the mucus and underlying intestinal mucosal layer in an in vitro model.
Submitted: January 28, 2014, Revised: August 27, 2014, Accepted: October 1, 2014. From the Departments of Surgery (M.E.D., L.N.D., D.M.L.) and Chemical Engineering (C.W.M.), Wayne State University, Detroit, Michigan. This study was presented at the Western Trauma Association meeting, March 2Y7, 2014, in Steamboat Springs, Colorado. Address for reprints: Lawrence N. Diebel, MD, Department of Surgery, 6C-University Health Center, 4201 Saint Antoine, Detroit, MI 48201; email: [email protected].
MATERIALS AND METHODS
DOI: 10.1097/TA.0000000000000499
IECs (NonYMucus-Producing Clone) HT29 cells, isolated from a colorectal adenocarcinoma from an adult female, were obtained from American Type Culture Collection and routinely cultured with Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum, 4.5-g/L glucose, and gentamicin in an atmosphere of 5% CO2 J Trauma Acute Care Surg Volume 78, Number 1
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at 37-C. Cells (5 105) were seeded on the apical surface of a polycarbonate membrane (3.0-Km pore size) (Transwell, Corning Costar Core, Cambridge, MA) in a two-chamber cell culture system and allowed to form polarized monolayers. Monolayer integrity was monitored by serial measurement of the transepithelial electrical resistance with a Millicell electrical resistance meter (Millipore Corp., Bedford, MA). When grown in this system, HT29 cells form polarized monolayers that are confluent after 10 days to 14 days in culture.
HT29-MTX Cells (Mucus-Producing Clone) The HT29-MTX-E12 cell line was obtained from HPA Cultures (Salisbury, United Kingdom) and routinely cultured with Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum, 4.5-g/L glucose, 1% nonessential amino acids, and 1% antibiotic/antimycotic (Gibco, Grand Island, NY) in an atmosphere of 5% CO2 at 37-C. Cells (5 105/mL) were seeded on the apical surface of a polycarbonate membrane (3.0-Km pore size) as described for HT29 cells earlier. This subpopulation of HT29 cells were differentiated into columnar absorptive cells and mucus secreting cells.17
Experimental Design HT29 and HT29-MTX IECs were grown to confluence in tissue culture flasks in the presence or absence of 90-pg/mL estrogen for up to 3 days. The cells were subsequently trypsinized and seeded onto two-chamber cell culture plates and grown to confluence. The nonadherent and adherent mucus layers were isolated, and mucus content was quantified by enzyme-linked immunosorbent assay (ELISA) specific for mucin 1 (MUC 1). In other experiments, the effect of estrogen on the viscosity and elasticity of the mucus layer was analyzed using rheologic testing. In a separate set of experiments, exposure of the IEC to 250-KM hydrogen peroxide (H2O2) for 30 minutes was introduced to assess the ability of estrogen to protect the intestinal barrier against oxidant-related injury. IEC apoptosis and permeability after H2O2 exposure were quantified by flow cytometry. Oxidative damage to IEC monolayers was measured by lipid hydroperoxide release and protein carbonyl content.
O-Linked Oligosaccharide Quantification Nonadherent mucin quantification was determined using a modification of the assay developed by Tanabe et al.18 Briefly, HT29 and HT-29 MTX cell monolayers and those treated with 90-pg/mL estrogen for 3 days were washed with phosphatebuffered saline (PBS) several times. These washings were then centrifuged at 10,000 G for 30 minutes, and the supernatants were removed. Ice cold ethanol at 60% by volume was added, and the samples were allowed to precipitate. The mucin fraction was recovered by centrifugation at 1,400 G for 10 minutes and finally dissolved in 5-mL dH20. The nonadherent mucin content was finally measured via fluorescence at 383 nm. Standard solutions of N-acetylgalactosamine (Sigma, St. Louis, MO) were used to calculate the amount of O-linked oligosaccharide chains liberated from mucins during the procedure.
Alcian Blue Quantification of Mucins Determination of the total amount of adherent mucin on the surface of HT29-MTX cells was quantitated using methods
similar to those outlined by Scott and Dorling19 and Scott and Willett.20 Briefly, HT-29 MTX cells with or without estrogen and washed free of nonadherent mucin with PBS were mixed with 1 mL of Alcian blue reagent (0.5% alcian blue in 50-mM sodium acetate containing 50-mM MgCl2). This mixture incubates for 2 hours at room temperature, after which the alcian blueYmucus complex was centrifuged at 2,000 G for 15 minutes. Once the precipitate is dissolved, the adherent mucus content was determined using an ELISA specific for MUC 1. Standards were prepared using chondroitin sulfate over the range of 1 Kmol to 10 Kmol.
MUC 1 ELISA MUC 1 content was quantitated in both the nonadherent and adherent mucin collected from HT-29 MTX cells using an ELISA assay kit specific for MUC 1 purchased from USCNK Life Science, Inc. (Wutnan, China). Standards and samples are run according to the kit instructions, and the amount of MUC 1 present in the samples is determined by comparing the optical density of the samples to the standard curve measured at 450 nm.
Rheology Testing To evaluate the viscoelastic properties of HT29 and HT29-MTX cells exposed to 90 pg/mL estrogen (E2) for 3 days, the IECs were grown on glass cover slips (2.5-cm circles) in 6-well plates (Fisher Scientific, Hanover Park, IL) at a concentration of 1 105 cells/mL. Once cells reach confluence on the cover slips, they are mounted with super glue (cyanoacrylate adhesive) directly to the lower platen of a Rheometrics ARES rheometer equipped with 2.5-cm parallel plates. The cell culture samples are then tested in small amplitude oscillatory shear flow to measure the storage modulus G’ (U) and loss modulus G’’ (U), which characterize the linear viscoelastic response of the fluid layer. All rheologic testing is conducted at a temperature of 37-C, controlled by a humidified environmental chamber, and at 0.5% strain, which assures that the mucus layer response remains in the linear viscoelastic regimen. Each sample is tested at oscillatory frequencies ranging from 0.1 rad/s to 100 rad/s (0.016Y16 Hz). Variation of viscoelasticity with position within the mucus layer causes a nonuniform strain within the mucus layer. Therefore, the measured G’ and G’’ values must be interpreted as apparent, rather than absolute, values of these viscoelastic material functions. Viscosity measurements of mucus samples washed from the surface of HT29 and HT29-MTX cell cultures were conducted with a Contraves LS-30 rheometer at 13 per second, using cup-and-bob fixtures.
Determination of Lipid Peroxide Malondialdehyde (MDA), a byproduct of lipid peroxide, was detected in HT29-MTX cells using an MDA Adduct ELISA Kit (Cell Biolabs, Inc., San Diego, CA). The kit was used according to manufacturer’s instruction and has a sensitivity limit of 2-pmol/mg MDA Adduct.
Determination of Protein Carbonyl Content Protein carbonyl content in HT29-MTX cells was calculated using a Protein Carbonyl Spectrophotometric Assay Kit (Cell Biolabs, Inc.). Cell samples were run according to the
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manufacturer’s instructions, and the protein carbonyl content was calculated. Data are expressed as nanomole per milligram.
Measurement of Reactive Nitrogen Intermediates Nitration of tyrosine residues, a marker of reactive nitrogen intermediate (RNI)Ymediated injury, was measured using the OxiSelect Nitrotyrosine ELISA kit (Cell Biolabs Inc.). Mucus samples were added according to the kit instructions, and nitrated bovine serum albumin (BSA) standards were also plated to provide a known nitrated BSA standard curve. The protein nitrotyrosine content in the samples is determined by comparison with the nitrated BSA standards provided.
Apoptosis Confluent HT29-MTX cells from the different treatment groups were analyzed for apoptosis using the TACS Annexin-V Apoptosis Detection Kit (R&D Systems, Minneapolis, MN). HT29-MTX cells were incubated with an Annexin-V Incubation reagent for 15 minutes in the dark then mixed with a 1 binding buffer and analyzed by flow cytometry within 1 hour for maximal signal. Results were expressed as percent apoptosis compared with control cells.
Permeability Determination Using Fluorescein IsothiocyanateYDextran Confluent HT29-MTX cells with or without estrogen grown on inserts were exposed to 250-KM H2O2 for up to 30 minutes. Fluorescein isothiocyanate (FITC)Ydextran (4,000 molecular weight, 2.2 mg/mL in PBS) was added to each insert, and the monolayer was incubated for 2 hours at 37-C. Monolayer permeability to FITC-dextran was then determined by measuring the fluorescence of the basal solution in each well at 485 nm (excitation wavelength) and 535 nm (emission wavelength). Permeability data were expressed as nanomole per square centimeter per hour.
Statistical Analysis An analysis of variance with a post hoc Tukey test was used to analyze the data. Statistical significance was inferred at p values of less than 0.001. All data are expressed as mean (SD).
RESULTS The effect of treatment of HT29-MTX cells with estrogen led to significant increases in the mucin content of the mucus layer (Fig. 1A and B). There were 45% to 47% increases in the mucin content in the nonadherent and adherent mucus layers, respectively (p G 0.001 vs. no estrogen HT29-MTX group). The impact of estrogen treatment on the viscoelastic properties of the mucus layer are shown in Figure 2A and B. Estrogen treatment resulted in a twofold increase in viscosity and a nearly eightfold increase in elasticity of the mucus layer (p G 0.001 vs. no estrogen HT29-MTX group). The data shown in Figure 2A and B reflect a shear stress typical of the intestine in a fasting state. The protective effect of estrogen on oxygen radical damage to the mucus layer is demonstrated in Figure 3. Both protein oxidation (protein carbonyl) and lipid oxidation (lipid hydroperoxide) products were determined. Exposure to H2O2 led to a five times increase in lipid hydroperoxide and a three times increase in protein carbonyl content (Fig. 3A, p G 0.001 vs. 96
Figure 1. (A), The effect of estrogen pretreatment on nonadherent mucin content in HT29-MTX cells. The nonadherent mucus layer was isolated from HT29-MTX cells with or without 90-pg/mL estrogen for up to 3 days. The nonadherent component of the mucus layer was measured using fluorescence at 383 nm. Known N-acetylgalactosamine standards were used to quantify O-linked oligosaccharide chains liberated from the mucin layer. (B), The effect of estrogen pretreatment on adherent mucus content of HT29-MTX cells. The adherent mucus layer was isolated from HT29-MTX cells with or without 90-pg/mL estrogen for up to 3 days. The protein MUC 1 was quantified by ELISA with an antibody specific for the MUC 1 protein.
HT29-MTX without H2O2 treatment). Pretreatment with estrogen resulted in the significant reduction of both of these oxidant-related markers to near-baseline levels (p G 0.001 vs. HT29-MTX + H2O2). RNIs were increased nearly two times in HT29-MTX cells treated with H2O2 (Fig. 3B, p G 0.001). However, these levels were reduced to RNI concentrations of nonYH2O2-treated HT29-MTX cells. The effect of luminal H2O2 challenge on the underlying HT29 monolayer was indexed by apoptosis and permeability (Figs. 4 and 5). There was a greater than three times increase in HT29-MTX apoptosis and a twofold increase in monolayer permeability after exposure to H2O2. Estrogen pretreatment resulted in marked reductions in apoptosis and permeability noted after H2O2 challenge (p G 0.001 vs. HT29-MTX + H2O2).
DISCUSSION There is increasing recognition of the role of the mucus layer covering the gastrointestinal epithelium in protection * 2015 Wolters Kluwer Health, Inc. All rights reserved.
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of the mucus layer after H2O2 challenge. In this regard, Brownlee et al.14 failed to demonstrate any changes in the absolute value of storage and loss moduli of colonic mucus after reactive oxygen species (ROS) treatment (50-nM H2O2). However, because mucus is not a homogeneous fluid, the macroscopic viscoelastic measurements are an average assessment of the rheology of nanoscopically heterogeneous environments of the mucus layer. Thus, recent studies have focused on the importance of mucus microrheology to better understand the biophysical properties of mucus.22 Previous studies have shown that estrogen protects against T/HS intestinal injury and remote organ failure.3 This may in part be related to a cytoprotective effect by activation of functional estrogen receptors on IEC.23 In another study, Sheth et al. demonstrated that there was preservation of the intestinal mucus layer in females and postulated that this may have a
Figure 2. (A), The effect of estrogen pretreatment on mucus layer viscosity in HT29-MTX cells. HT29 and HT29-MTX cell monolayers and those treated with 90-pg/mL estrogen for 3 days were washed with PBS several times to remove the mucus layer. The washings were subjected to rheologic testing using a Contraves LS-30 rheometer. Viscosity is expressed as centipoises (Cp) at a shear rate of 13 per second. (B), Analysis of the elasticity of the mucus layer in HT29-MTX cells pretreated with estrogen. HT29 and HT29-MTX cells exposed to 90-pg/mL estrogen (E2) for 3 days were grown on glass cover slips and mucus storage modulus (G’) measured using the ARES rheometer. G’ values, representing the elastic response of the mucus layer, were measured at a frequency of 10 rad/s. and 1% strain at a position 75 Km above the substrate surface.
against gastric acid, pancreatic digestive enzymes, and luminal bacteria especially under stress conditions.15,16,21 Mucins are highly glycosylated proteins and are the major organic component of mucus.9,10 The viscoelastic properties of mucus are primarily due to mucin/water content and secondarily to lipids, other proteins, and iron content.6 The importance of mucin concentration on mucus viscoelasticity is illustrated by cervical mucus.6 The mucin concentration in nonovulatory mucus has been noted to increase by only twofold or fourfold; however, this markedly increased its viscoelasticity, making it virtually impenetrable to sperm. In our study, exposure to physiologic concentrations of estrogen increased mucin content of the mucus layer and led to marked changes in the rheologic properties of the mucus layer. In our study, the elastic component of the viscoelastic shear modulus (G’) and the viscous component (G’’) were increased by estrogen treatment. As a non-Newtonian fluid, this varied with the shear stress but was apparent over a wide range of the shear stress applied. We did not obtain rheologic measurements
Figure 3. (A), The effect of H2O2 on oxidative damage to the mucus layer of IEC monolayers: measurement of lipid hydroperoxide and protein carbonyl content. A by-product of lipid peroxide, MDA, was measured in the mucus layer collected from HT29-MTX cells using an MDA Adduct ELISA Kit. Protein carbonyl content was measured in the mucus layer of HT29-MTX cells exposed to 90-pg/mL estrogen for 3 days and 250-KM H2O2 for up to 30 minutes using a Protein Carbonyl Spectrophotometric Assay Kit. (B), The effect of H2O2 on oxidative damage to the mucus layer of IEC monolayers: measurement of RNIs. Oxidative damage mediated by RNI increased in the mucus layer isolated from HT29-MTX cells exposed to 250-KM H2O2 for 30 minutes. Pretreatment of HT29-MTX cells with 90-pg/mL estrogen for 3 days reduced the damage from RNI in the mucus layer.
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Figure 4. The protective effect of estrogen on apoptosis after H2O2 treatment of HT29-MTX cells. HT29-MTX cells were cocultured with 90-pg/mL estrogen for 3 days followed by exposure to 250-KM H2O2 for 30 minutes. Apoptosis was quantified using flow cytometry.
protective effect against shock-induced gut injury and subsequent remote organ injury. In our study, estrogen treatment of HT29 cells led to changes in the rheologic properties of the mucus layer and increased mucus content. Oxidant injury of the intestinal mucus layer may be important in the pathogenesis of T/HS gut barrier failure.24 Because of the protective effects of estrogen on T/HS gut barrier failure and the physiochemical changes in the mucus layer noted in our study, we studied the effect of a standard oxidant challenge on the mucus layer and intestinal barrier function. Our studies demonstrated significantly decreased protein carbonyl, nitrated tyrosine residues, and lipid peroxidation in HT29-MTX cells treated with estrogen and then exposed to H2O2. This may reflect enhanced antioxidant properties of the mucus layer because of increased mucin content. Other possibilities include other genomic and nongenomic effects of estrogen on IECs and/or mucus secreting cells. Native mucus gel has been shown to be more resistant to ROS damage than purified mucin.14 Thus, nonmucin components of the mucus may also have ROS-scavenging properties.
Figure 5. Estrogens effect on IEC monolayer permeability after H2O2 treatment. Monolayer permeability to FITC-dextran (FD4) was determined after HT29-MTX cells were exposed to 90-pg/mL estrogen for 3 days and 250-KM H2O2 for up to 30 minutes. 98
Although lipids such as phosphatidylcholine make up only a small percentage of intestinal mucus, they may be important in the protection against oxygen radical damage to mucin. Of note, most of the lipids in mucus is associated with hydrophobic domains of the mucin glycoproteins.6 Exogenous H2O2 was used to provide a uniform oxidant challenge to the mucus layer and the IECs in our model. Endogenous sources of oxyradicals following gut perfusion injury in vitro include IEC xanthine oxidase and phospholipase A2.25,26 Of note, the action of phospholipase A2 on the lipid component of the mucus layer (phosphatidylcholine) may increase oxygen radical damage to mucins as lipid binding to mucin has a protective effect against oxygen radicals.13 Additional studies on the effect of estrogen on the lipid component of the mucus layer before and after oxygen challenge are therefore warranted. In summary, estrogen treatment increased mucin content and enhanced the rheologic properties of the mucus layer of HT29-MTX cells. This afforded protection to the mucus layer and the underlying intestinal epithelial monolayer following oxidant challenge. The protective effect of estrogen against oxidative stress likely includes genomic and nongenomic pathways.1 Our study suggests that this in part is due to enhanced rheologic and oxygen scavenging properties of the mucus layer following treatment with estrogen. AUTHORSHIP L.N.D., D.M.L., M.E.D., and C.W.M. designed this study. L.N.D., D.M.L., M.E.D., and C.W.M. performed the data collection, data analysis, statistical analysis, and data interpretation. L.N.D., D.M.L., M.E.D., and C.W.M. prepared the figures. L.N.D., D.M.L., M.E.D., and C.W.M. wrote and critically revised the article. L.N.D., D.M.L., M.E.D., and C.W.M. claim overall responsibility.
DISCLOSURE The authors declare no conflicts of interest.
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