Attenuating effect of taurine on lipopolysaccharide-induced acute lung injury in hamsters

Pharmacological Research 60 (2009) 418–428

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Attenuating effect of taurine on lipopolysaccharide-induced acute lung injury in hamsters Tapan M. Bhavsar, Jerome O. Cantor, Sanket N. Patel, Cesar A. Lau-Cam ∗ Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John’s University, Jamaica, NY 11439, USA

a r t i c l e

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Article history: Received 20 April 2009 Received in revised form 16 May 2009 Accepted 16 May 2009 Keywords: Lipopolysaccharide Taurine Hamsters Lung Inflammation Oxidative stress Apoptosis

a b s t r a c t This study has evaluated the ability of the semiessential amino acid taurine to attenuate lipopolysaccharide (LPS)-induced lung inflammation, oxidative stress and apoptosis in a small animal model. For this purpose, bacterial LPS (0.02 mg in phosphate buffered saline (PBS) pH 7.4) was instilled intratracheally into female Golden Syrian hamsters, before or after a 3-day intraperitoneal treatment with a single dose (50 mg/kg in PBS pH 7.4) of taurine. At 24 h after the last treatment, lung tissue and bronchoalveolar lavage fluid (BALF) samples were collected. In comparison to samples from animals receiving only PBS pH 7.4, serving as controls, those of LPS-stimulated animals exhibited a higher count of both total leukocytes and neutrophils in the BALF, and increased incidence of apoptosis, depletion of intracellular glutathione and evidence of inflammation confined to the parenchyma in the lung. In addition, LPS caused cells in the BALF to exhibit a higher expression of tumor necrosis factor-1, a higher activity of caspase-3, marked lipid peroxidation, and altered activities of catalase, glutathione peroxidase and superoxide dismutase relative to control samples. In contrast, a treatment with taurine was found to significantly attenuate all of the cellular and biochemical alterations induced by LPS, more so when given before rather than after the endotoxin. The present results suggest that taurine possesses intrinsic antiinflammatory and antioxidant properties that may be of benefit against the deleterious actions of LPS in the lung. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Acute lung injury (ALI) is a common clinical complication of infection by Gram negative bacteria and an important cause of morbidity and mortality among humans [1]. The LPS component present in the outer membrane of such bacteria is regarded as a major contributor to ALI [2,3] and, in high doses, to the later development of respiratory distress syndrome [4]. LPS-induced ALI is characterized by the infiltration of neutrophils and macrophages into alveoli [4–9], the release of chemokines and proinflammatory cytokines from activated macrophages and neutrophils [4,5,10], the expression of hemoxygenase-1 [10], alveolitis [9,11], acute inflammatory airway response [6], and apoptosis in bronchoalveolar cells [5,10,12]. In addition, the excessive accumulation of inflammatory mediators, especially cytokines, chemokines, adhesion molecules, and bioactive lipid products [12,13] contribute to increased pulmonary vascular permeability, interstitial edema [11,14,15] and eventual cell death [16]. Furthermore, endotoxin-induced ALI is associated with an exaggerated production of cell-damaging reactive oxygen species (ROS), including superoxide anion radical, hydroxyl radical

∗ Corresponding author. Tel.: +1 718 990 6023. E-mail address: [email protected] (C.A. Lau-Cam). 1043-6618/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2009.05.006

and hydrogen peroxide, by neutrophils sequestered within the lung vasculature and by stimulated macrophages [8,10,17], a situation that culminates in lipid peroxidation [8], altered antioxidant enzyme activity [8,10], and the release of phagocyte-derived nitric oxide into plasma [15] and bronchoalveolar lavage fluid [18]. On the premise that inhibition of ROS formation by antioxidant compounds will preclude endotoxin-induced cellular damage [14,19], various natural [14,17–19], semisynthetic [11] and synthetic [13,15] compounds with demonstrable antioxidant properties have been tested as protectants against LPS-induced lung injury. One of the compounds that have been extensively tested for this purpose is taurine, a ubiquitous nonprotein ␤-aminosulfonic acid with known antioxidant properties [20–23] and regulatory action on the release of proinflammatory cytokines in humans and experimental animals [24]. Although earlier work from other laboratories have suggested taurine to be protective against lung damage in various models of inflammatory lung injury [20–23], studies directed at evaluating this sulfur-containing amino acid for its effects on the biochemical and pathophysiological events associated with LPS-induced ALI appear to be rather limited. For example, there are in vitro studies that have assessed the effects of taurine on the antibactericidal and antiinflammatory functions of neutrophils following an exposure to endotoxin [25], and the role of this amino acid as a modulator of cell death, intracellular calcium levels and oxidative state in response to LPS and tumor necrosis factor-␣ in human endothelial cells [26].

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In terms of in vivo studies, taurine have been examined for the ability to normalize hemodynamic changes, neutropenia, neutrophil respiratory burst activity and pulmonary myeloperoxidase activity accompanying endotoxin-related lung injury in sheep [29]; and to counteract respiratory burst activity and function of neutrophils from guinea pig treated with the endotoxin [28]. The present study was undertaken in hamsters to examine the ability of taurine to attenuate cellular infiltration, lipid peroxidation, apoptotic cell death and histological changes in the lungs as a result of an intratracheal treatment with bacterial LPS. Another objective of the study was to determine whether the order of administration of taurine and LPS influences the magnitude of the effects of taurine on LPS-induced alterations. 2. Materials and methods 2.1. Materials All the chemicals and reagents used in the study were purchased from local commercial sources. Hydrogen peroxide (H2 O2 , 30%, w/w), lipopolysaccharide (LPS, serotype: O26:B6 obtained from American Type Culture Collection no. 12795; with short chain-length approximating that of mutant rough strain LPS), phosphate buffer saline (PBS), reduced glutathione (GSH), taurine, trichloroacetic acid (TCA), sulfosalicylic acid (SSA), 1,1,3,3tetraethoxypropane (TEP) and thiobarbituric acid (TBA), were from Sigma–Aldrich, St. Louis, MO; 0.1 N and 6 N hydrochloric acid (HCl) were from Mallinckrodt Baker, Inc., Phillipsburg, NJ; and meta-phosphoric acid (MPA) was from Aldrich Chemical Co., Inc., Milwaukee, WI. 2.2. Animals A group of 120 female, Golden Syrian hamsters, 5–6 weeks old, weighing between 100 ± 15 g, was used in all the experiments (Harlan, Indianapolis, IN). The animals were maintained on a standard hamster chow and filtered tap water on an ad libitum basis, and were kept in a temperature-controlled environment (21 ± 1 ◦ C) with an alternating 12 h light–12 h dark cycle. The study received the approval of the Institutional Animal Care and Use Committee of St. John’s University, and the animals were cared according to guidelines established by the United States Department of Agriculture. 2.3. Treatments with LPS and taurine To determine the effects of a pretreatment with taurine on LPS-induced lung injury, hamsters were treated with taurine (as a solution in phosphate buffered saline (PBS) pH 7.4, 50 mg/kg/0.5 ml/day) by the intraperitoneal (i.p.) route for 3 days, followed successively by an i.p. dose of pentobarbital sodium (90 mg/kg/0.4 ml) to induce anesthesia, and an intratracheal (i.t.) instillation of LPS (0.2 ml of 0.1 mg/ml in PBS pH 7.4) on day 4. Controls were treated with: (a) i.p. PBS pH 7.4 for 3 days followed by i.t. LPS on day 4 (positive control), and (b) only i.t. PBS pH 7.4 on day 4 (negative control). To determine the effects of a posttreatment with taurine on LPS-induced lung injury, hamsters received LPS (0.2 ml of 0.1 mg/ml in PBS pH 7.4) by i.t. instillation on day 1, followed by taurine (as a solution in phosphate buffered saline (PBS) pH 7.4, 50 mg/kg/0.5 ml/day) by i.p. route for 3 days. Control animals were treated with (a) i.t. LPS on day 1 followed by i.p. PBS pH 7.4 on days 2–4 (positive control), and (b) only with i.t. PBS pH 7.4 (negative control). All i.t. instillations were carried out using a 1-ml syringe fitted with a 27-gauge needle. Following an i.t. delivery, the incision was closed with metal clips.

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On day 5, 24 h after a LPS instillation or a taurine treatment, the animals were sacrificed using a high dose of pentobarbital sodium (240 mg/kg/0.7 ml, i.p.), and bronchoalveolar lavage fluid (BALF) samples were collected by rinsing the bronchoalveolar surface with one 4 ml and two 3 ml volumes of PBS pH 7.4, and bringing the volume of the pooled washings to 10 ml with additional PBS pH 7.4. Immediately thereafter, the lungs were surgically removed, washed without delay with ice-cold physiologic saline, patted dry with filter paper, frozen in liquid nitrogen, and kept at −20 ◦ C until used in an assay. The occurrence of pulmonary inflammation was established by both histopathological examination of lung tissue and the measurement of the number of neutrophils and tumor necrosis factor receptor-1 (TNFR1) positive macrophages in BALF. Evidence of cell death was ascertained by verifying alveolar septal cell apoptosis in lung tissue by a TUNEL assay and by measuring the activity of caspase-3 in BALF. The development and extent of oxidative stress was determined by measuring the levels of lung reduced glutathione (GSH) and the assay of malondialdehyde (MDA) and the activities of catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) in BALF. 2.4. Histological studies Following euthanasia of the animals with a high dose of pentobarbital (240 mg/kg/0.7 ml, i.p.), the lungs were fixed in situ for about 2 h with 10% neutral-buffered formalin at a pressure of 20 cm of water. The lungs, along with the attached heart, were surgically removed through a vertical incision along the thorax, and fixed in 10% neutral-buffered formalin for an additional 48 h. After excising any extrapulmonary structures with the help of a scalpel, the lungs were cut into pieces of a size suitable for histological processing, put though a standard embedding procedure in paraffin, sectioned on a microtome, and stained with hematoxylin and eosin (H&E). The sections where then examined for evidence of inflammation with the help of a light microscope. 2.5. Determination of lung injury The presence of lung injury in the tissue sections was graded using the scale of Szarka et al. [29]. The following scoring values were used: 0 = no reaction in the alveolar walls, 1 = diffuse reaction in the alveolar walls but without thickening of the interstitium, 2 = diffuse presence of inflammatory cells in the alveolar walls with a slight thickening of the interstitium, 3 = moderate interstitial thickening accompanied by inflammatory cell infiltrates, and 4 = interstitial thickening involving more than one-half of the microscopic field. Results were expressed as the average of the values from 50 microscopic fields. 2.6. Collection of BALF Following euthanasia with i.p. pentobarbital, lungs were rinsed three times with normal saline (once with 4 ml, twice with 3 ml aliquot). The rinsings were pooled together, centrifuged at 1000 × g for 5 min, and the pelleted cells resuspended in 1.0 ml of PBS pH 7.4. The total leukocyte count was determined with a hemocytometer. Differential counts were obtained on cytospin preparations treated with Wright’s stain. 2.7. Immunocytochemistry of TNFR1 Cytospin preparations were fixed with ethanol and permeated with an alcohol–acetic acid (2:1) mixture. Endogenous peroxidase activity was quenched with 0.3% H2 O2 and nonspecific binding was

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blocked with goat serum (Vector Laboratories, Inc., Burlingame, CA). After an incubation with anti-mouse TNFR1 polyclonal antibody (Stressgen, Victoria, BC) at room temperature for 30 min, and a rinsing with PBS pH 7.4, the sample was successively incubated with biotinylated goat anti-rabbit antibody or rabbit anti-rat antibody (Stressgen, Victoria, BC) at room temperature for 1 h, and with ABC reagent (Vector Laboratories, Inc., Burlingame, VT) for 1 h, prior to staining with 3,3 -diaminobenzidine substrate in the dark, and counterstaining with methyl green. The slides were examined under a light microscope at 400× magnification for the presence of macrophages stained in brown. For comparative purposes, a negative control sample that had not been incubated with TNFR1 primary antiserum was also examined. The results were expressed as the number of macrophages staining for TNFR1 in a group of 200 cells counted in the same section. The number of macrophages staining for TNFR1 was reported as a percentage of the total number of macrophages examined. 2.8. TUNEL assay for apoptosis Tissue sections were digested with 20 ␮g/ml of proteinase K (Sigma–Aldrich, St. Louis, MO) at room temperature, washed with distilled water, and treated with 0.3% H2 O2 in PBS pH 7.4 to quench endogenous peroxidase. After incubation with TdT enzyme (Chemicon International, Temecula, CA) at 37 ◦ C for 1 h, the samples were exposed to anti-digoxigenin conjugate (Chemicon International, Temecula, CA) at room temperature for 30 min. The samples were stained with DAB peroxidase substrate (Vector Laboratories, Inc., Burlingame, VT), counterstained with methyl green and examined under a microscope. Twenty-five high-power (HPF, 400× magnification) microscopic fields were examined, and the results were expressed as the mean number of TUNEL-positive cells per HPF. 2.9. Caspase-3 assay The BALF samples were centrifuged in a conical tube at 250 × g for 10 min. After discarding the supernatant, the pelleted cells were used for the assay of caspase-3 activity using a commercial colorimetric assay kit (R&D Systems, Minneapolis, MN) on a 96-well EIA plate (Corning Inc., Corning, NY). For this purpose, a 100 ␮l aliquot of the pellet was treated with 50 ␮l of cell lysis buffer, followed by incubation of the lysate on an ice bath for 10 min, and centrifugation of the suspension at 10,000 × g for 1 min. Then, a. mixture of 50 ␮l aliquot of cell lysate with 50 ␮l of 2× Reaction Buffer 3 was treated with 10 ␮l of DTT solution (1.0 M DTT stock/ml of 2× Reaction Buffer 3), followed by 5 ␮l of 4 mM DEVD-pNA substrate, and allowed to react at 37 ◦ C for 2 h in the dark. A negative control was prepared to contain the same components as the sample preparation but omitting the DEVD-pNA substrate; and the cell lysis buffer served as a blank preparation. The caspase-3 activity, derived from the absorbance value of the sample preparation read with a plate reader set at 405 nm, was expressed as absorbance U/␮g of protein. 2.10. Preparation of tissue homogenates Following their removal, the lung samples were rinsed immediately with physiological saline, patted dry with filter paper, weighed, and perfused with ice-cold physiologic saline. A portion of lung sample was minced with a razor blade, and homogenized in PBS pH 7.4 using hand held tissue homogenizer (Tissue-Tearor® , BioSpec Products, Inc., Bartlesville, OK). The homogenate was sonicated for 1.5 min using 30 s pulses followed by 30 s pauses while kept cold with the help of an ice bath. The suspension was cen-

trifuged at 14,000 rpm for 30 min, and the supernatant used for the assay of GSH. 2.11. CAT activity assay The activity of CAT was measured as described by Aebi [30]. A 0.1 ml aliquot of BALF was mixed with 1 ml of PBS pH 7.4 and 1 ml of 30 mM H2 O2 in a spectrophotometric quartz cuvette, and the absorbance of the reaction mixture read without delay at 240 nm twice, immediately after mixing and 1 min later. The activity of CAT, in U/mg of protein, was calculated from the equation [OD × Vc × df/(0.071 × Vs)], where OD is the difference between the first and second absorbance readings; Vc is the volume of the spectrophotometric cuvette in ml; Vs is the volume of sample taken in ml; df is the dilution factor; and 0.071 is the molar extinction coefficient of H2 O2 . 2.12. GPx activity assay The activity of GPx was measured indirectly by a coupled reaction with glutathione reductase using the spectrophotometric method of Günzler and Flohé [31]. A 0.1 ml aliquot of BALF was mixed with 0.1 ml each of solutions containing 54 U/ml glutathione reductase, 10 mM GSH, and 15 mM ␤-NADPH in PBS pH 8.0. After standing at room temperature for 15 min, 0.1 ml of 3 mM H2 O2 was added to the reaction mixture, and the change in absorbance at 340 nm was measured for 1 min. The results are expressed in U/ml of BALF/min. 2.13. SOD activity assay This enzyme activity was determined based on the inhibitory action of SOD on the reduction of nitroblue tetrazolium (NBT) by superoxide anion generated by a xanthine-xanthine oxidase system and modifying the conditions described by Ukeda et al. [32]. To a 0.1 ml aliquot of BALF, 2.5 ml of PBS pH 8.0 containing 15% BSA, and 0.1 ml each of solutions containing 3 mM xanthine, 3 mM EDTA, 0.75 mM NBT, and 56 U/ml xanthine oxidase were added. After allowing the reaction mixture to stand at room temperature for 30 min, its absorbance was read on a spectrophotometer at 560 nm. The enzyme activity was expressed as U/ml of BALF. 2.14. Thiobarbituric acid reactive substances assay The concentration of MDA in the BALF was measured as thiobarbituric acid reactive substances (TBARS) using the method of Buege and Aust [33]. To this effect, a 0.1 ml aliquot of BALF was mixed with 0.9 ml of a reagent containing 15% TCA (w/v)–0.375% TBA (w/v)–0.25 N HCl, and heated at 90 ◦ C for 1 h. After allowing the mixture to cool to room temperature, it was centrifuged at 2000 × g for 5 min to remove insolubles, and its absorbance read at 535 nm on a spectrophotometer. The level of MDA was derived from a standard curve prepared from serial dilutions of a 3.2 ␮M stock solution of 1,1,3,3-tetraethoxypropane that were treated in identical manner as the BALF samples. The concentration of MDA was expressed as nmol/mg of protein. 2.15. GSH assay The concentration of reduced glutathione (GSH) in the lung homogenate was measured following its reaction with 5,5 dithiobis (2-nitrobenzoic acid) (DTNB) according to Ellman [34]. A 100 ␮l aliquot of lung homogenate was mixed with 100 ␮l of 5% meta-phosphoric acid, and the mixture centrifuged at 2000 × g for 5 min. A 100 ␮l portion of the clear supernatant was mixed

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3. Results 3.1. Effects of LPS and taurine-LPS on lung histology Treating hamsters with taurine for 3 days, either before or after LPS administration, resulted in significant attenuation of the lung injury caused by LPS, with the attenuation being greater when taurine was given before rather than after LPS. Thus, the mean inflammatory index of animals receiving taurine before LPS was ∼40% lower than that of animal receiving LPS alone (1.8 vs. 3.0, P < 0.001, Fig. 1A), an effect that was reduced to ∼28% when taurine was given after LPS (1.8 vs. 2.5, P < 0.01, Fig. 1B). In both instances, the inflammatory response appeared to be limited to the parenchymal tissue and not to involve the airways. The mean inflammatory index of animals receiving only PBS (0.2) probably reflects a transient inflammatory response brought about by the intratracheal instillation procedure itself (Fig. 2). 3.2. Effects of LPS and taurine-LPS on total leukocyte and neutrophil counts in BALF and lung Fig. 1. Animals receiving taurine either before (A) or after (B) a treatment with LPS showed a significant reduction in lung inflammation compared to animals treated with LPS alone. Each bar represents the mean ± S.E.M. for n = 6. *P < 0.001 vs. PBS + LPS and # P < 0.01 vs. LPS + PBS.

with 1.9 ml of 0.1 M phosphate buffer pH 8.0 and 20 ␮l of 0.02 M DTNB in 0.1 M phosphate buffer pH 8.0, and the absorbance of the resulting product read at 412 nm on a spectrophotometer. The concentration of GSH in the sample was derived by reference to a calibration curve of GSH prepared from serial dilutions of a 240 ␮M GSH stock solution that were treated in identical manner as the lung homogenate samples. The result was reported as ␮mol/mg of protein. 2.16. Statistical analysis of the data The experimental results were expressed as the mean ± S.E.M. Statistically significant differences among treatment groups was determined by Newman–Keuls multiple comparisons, with the difference being considered significant at P < 0.05.

A 3-day treatment with taurine, either before or after LPS administration, led to a significant lowering of the total number of leukocytes seen in BALF from animals treated with LPS alone, with the magnitude of the effect being much greater when taurine was administered before rather than after LPS. In animals receiving taurine ahead of LPS, the mean total number of leukocytes in BALF was found to be 72% lower than in BALF of animals on LPS alone (7.6 × 103 cells/ml vs. 27.0 × 103 cells/ml, respectively, P < 0.001). In contrast, a posttreatment with taurine lowered the mean total number of leukocytes in BALF from animals treated with LPS by only 44% relative to the total leukocytes count seen in the absence of taurine (14.5 × 103 cells/ml vs. 26.0 × 103 cells/ml, P < 0.001) (Fig. 3). The administration of taurine, either before or after LPS, was also able to significantly decrease the percentage of neutrophils in BALF, as seen in a differential cell count, from animals treated with LPS. The mean percentage value of neutrophils relative to control was 80.6% (P < 0.001) in the BALF of animals receiving taurine before LPS and 95.1% (P < 0.001) in animals receiving LPS alone (Fig. 4). On the other hand, reversing the treatment order led to an insignificant loss

Fig. 2. Photomicrographs of sections from hamster lungs stained with H&E. (A)–(C) and (D)–(F) show the results for animals receiving taurine as a pretreatment and as a posttreatment to LPS, respectively. Sections from animals receiving only PBS pH 7.4 showed no inflammatory reaction in the alveolar wall (A and D). Sections from animals serving as positive controls, treated with PBS pH 7.4 for 3 days before (B) or after (E) an i.t. treatment with LPS (0.02 mg) showed a moderate inflammatory reaction and a mild thickening of the alveolar wall. The administration of taurine (50 mg/kg, i.p.) for 3 days before (C) or after (F) LPS showed minimal inflammatory reaction in the alveolar wall and no thickening of the interstitium relative to lung sections from animals treated with LPS alone (400× magnification).

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Fig. 3. Animals receiving taurine either before (A) or after (B) a treatment with LPS showed a significant reduction in total BALF leukocyte count compared to animals treated with LPS alone. Each bar represents the mean ± S.E.M. for n = 9. *P < 0.001 vs. PBS + LPS and # P < 0.001 vs. LPS + PBS.

Fig. 5. Animals receiving taurine either before (A) or after (B) a treatment with LPS showed a significant reduction in the total BAL neutrophil count compared to animals treated with LPS alone. Each bar represents the mean ± S.E.M. for n = 6. *P < 0.001 vs. PBS + LPS and # P < 0.001 vs. LPS + PBS.

in taurine potency, with the percentage of neutrophils averaging 84.8% in the LPS-taurine treated group and 95.6% in the LPS alone group (P < 0.001, Fig. 4). Taurine was also effective in lowering the total number of neutrophils infiltrating the lungs as a result of a treatment with LPS, with the extent of the effect depending on the order of the treatment. For example, dosing hamsters with taurine for 3 days prior to LPS led to a BALF mean total neutrophil count that was 84% lower (P < 0.001) than the mean total neutrophil count in the BALF of animals receiving only LPS (0.4 × 104 cells vs. 2.5 × 104 cells) (Figs. 5 and 6). In contrast, the mean total number of neutrophils in BALF from animals receiving taurine after LPS was decreased by only ∼55% (P < 0.001) relative to the number found in BALF from animals on LPS alone (1.2 × 104 cells vs. 2.6 × 104 cells, respectively) (Figs. 5 and 6).

3.3. Effects of LPS and taurine-LPS on TNFR1 expression by BALF cells The expression of TNFR1 on BALF cells induced by LPS was significantly decreased by a 3-day treatment with taurine regardless of the order of administration of taurine and LPS. Thus, while only about 25% of the cells in the BALF of animals receiving taurine before LPS were TNFR1-positive, about 60% were found to be TNFR1 positive in the BALF of animals receiving only LPS (P < 0.001, Figs. 7 and 8). Reversing the order of treatments reduced the attenuating effect of taurine on the number of cells expressing TNFR1 under the influence of LPS but, even then, the number was still significantly below that measured in BALF from animals treated only with LPS (∼39% vs. ∼64%, P < 0.001, Figs. 7 and 8). 3.4. Effects of LPS and taurine-LPS on the apoptosis of lung cells A 3-day treatment with taurine, either before or after LPS administration, had a significant decreasing effect on the number of BALF cells exhibiting apoptosis in comparison to cells from animals treated with LPS alone. The number of labeled alveolar cells per HPF was 0.4 in animals receiving taurine prior to LPS, and 0.8 in animals receiving LPS alone (P < 0.001, Figs. 9 and 10). Because of the limitations imposed by the procedure used to stain the alveolar cells, only those occupying the septa were amenable to examination. Although the counteracting effect of taurine against LPS-induced apoptosis was somewhat diminished when administered after a treatment with LPS (0.5 labeled cells per HPF), it was still significant relative to alveolar cells from animals receiving only LPS (0.8 labeled cells per HPF, P < 0.05, Figs. 9 and 10). 3.5. Effects of LPS and taurine-LPS on caspase-3 activity of BALF cells

Fig. 4. Animals receiving taurine either before (A) or after (B) a treatment with LPS showed a significant reduction in the percentage of BALF neutrophils, compared to animals treated with LPS alone. Each bar represents the mean ± S.E.M. for n = 9. *P < 0.01 vs. PBS + LPS and # P < 0.001 vs. LPS + PBS.

A 3-day treatment with taurine, either before or after one with LPS, caused a significant decrease in the caspase-3 activity of cells in BALF when compared to the activity of cells treated only with LPS, more so when given ahead of LPS rather than after (Fig. 11). While the mean absorbance value of an alveolar cell lysate at 405 nm was 0.009 U/␮g of protein in animals receiving taurine before a treatment with LPS, it was equal to 0.02 U/␮g of protein in animals on

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Fig. 6. Photomicrographs of cells in BALF from hamster lungs after staining with Wright solution. (A)–(C) and (D)–(F) show the results for animals receiving taurine as a pretreatment and as a posttreatment to LPS, respectively. Cells from animals receiving only PBS pH 7.4 showed a normal differential count, with the majority of cells being macrophages (A and D). BALF from animals serving as positive controls, treated with PBS pH 7.4 for 3 days before (B) or after (E) an i.t. treatment with LPS (0.02 mg) showed a significantly higher number of neutrophils and a few macrophages relative to animals on PBS pH 7.4 alone. The administration of taurine (50 mg/kg, i.p.) for 3 days before (C) or after (F) LPS showed a reduction in the number of neutrophils relative to BALF from animals receiving only LPS (magnification of 400×).

LPS alone (55% decrease, P < 0.01). Likewise, the mean absorbance value at 405 nm for samples from animals treated with taurine after a dosing with LPS was 0.01 U/␮g of protein and 0.02 U/␮g of protein in samples from animals receiving only LPS (50% decrease, P < 0.01, Fig. 11). 3.6. Effects of LPS and taurine-LPS on the formation of MDA by BALF cells A treatment with LPS led to 76% increase in the formation of MDA, measured as TBARS, in BALF cells relative to the value found

for control cells (P < 0.001). This effect was attenuated to a significant extent by a 3-day treatment with taurine, more so when taurine was given before than after LPS (Fig. 12). Thus, in animals receiving taurine as a pretreatment to LPS, the content of TBARS in BALF cells was 35% lower than in BALF cells from animals receiving only LPS (4.8 nmol/mg of protein vs. 7.4 nmol/mg of protein, respectively, P < 0.001). Reversing the order of the treatments led to only a 9% decrease relative in TBARS but which was still significant (6.8 nmol/mg of protein vs. 7.5 nmol/mg of protein, respectively, P < 0.01, Fig. 12). 3.7. Effects of LPS and taurine-LPS on the lung GSH level LPS caused a significant decrease in lung GSH (20%, P < 0.001) in comparison to the control value (Fig. 13). A 3-day treatment with taurine prior to LPS raised the lung GSH content by 18% relative to animals on LPS alone (47.1 ␮mol/mg of protein vs. 39.8 ␮mol/mg of protein, respectively, P < 0.001). In contrast, administering taurine for 3 days after a treatment with LPS was ineffective in reversing the decrease in GSH induced by LPS (39.2 ␮mol/mg of protein vs. 39.7 ␮mol/mg of protein, respectively, Fig. 13). 3.8. Effects of LPS and taurine-LPS on the activities of BALF antioxidant enzymes

Fig. 7. Animals receiving taurine either before (A) or after (B) a treatment with LPS showed a significant reduction in the number of TNFR1-positive macrophages compared to animals treated with LPS alone. Each bar represents the mean ± S.E.M. for n = 6. *P < 0.001 vs. PBS + LPS and # P < 0.001 vs. LPS + PBS.

The effects of taurine on LPS-induced changes in GPx, CAT and SOD activities of BALF cells were assessed on the basis of the results for sets of two experiments, and in which the only difference was the order of administration of taurine relative to that of LPS. Thus, LPS was found to markedly lower the CAT activity of BALF cells (by 52%, P < 0.001) relative to cells from control animals (Fig. 14). A 3day treatment with taurine, given either before or after LPS, raised the CAT activity above that seen with LPS alone, with the effect being greater when taurine was given before rather than after LPS (Fig. 14). Thus, giving taurine ahead of LPS doubled the CAT activity seen with BALF cells from animals receiving only LPS (0.04 U/mg of protein vs. 0.02 U/mg of protein, respectively, P < 0.001) (Fig. 14). In contrast, giving taurine as a posttreatment to LPS raised the CAT by

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Fig. 8. Photomicrographs of cells in BALF from hamster lungs after staining with Vectastain® Elite ABC kit. (A)–(C) and (D)–(F) show the results for animals receiving taurine as a pretreatment and as a posttreatment to LPS, respectively. BALF from animals receiving only PBS pH 7.4 lacked TNFR1 positive cells (A and D). BALF from animals serving as positive controls, treated with PBS pH 7.4 for 3 days before (B) or after (E) an i.t. treatment with LPS (0.02 mg), showed a modest increase in the number of TNFR1 positive macrophages relative to BALF from animals receiving only PBS pH 7.4 alone. The administration of taurine (50 mg/kg, i.p.) for 3 days before (C) or after (F) LPS showed a reduction in the number of TNFR1 positive macrophages relative to BALF from animals receiving LPS alone (magnification of 400×).

only 1.5-fold (0.03 U/mg of protein vs. 0.02 U/mg of protein, respectively, P < 0.01) (Fig. 14). The trend of the effects of LPS and of taurine-LPS on the SOD activity of BALF cells resembled those noted for the CAT activity. In this case, while LPS decreased the SOD activity by an average of about 29% relative to the control value (0.773 ± 0.060 U/ml vs. 1.095 ± 0.168 U/ml, respectively, P < 0.01, Fig. 15), a pretreatment with taurine raised the activity by about 1.95-fold over that with LPS alone (1.532 ± 0.180 U/ml vs. 0.786 ± 0.073 U/ml, respectively, P < 0.001, Fig. 15). On the other hand, a posttreatment with taurine raised the SOD activity by only about 1.28-fold over that seen with LPS alone (0.970 ± 0.019 U/ml vs. 0.760 ± 0.049 U/ml, respectively, P < 0.05, Fig. 15).

At variance with the results gathered for both CAT and SOD, those stemming from the effects of LPS and taurine-LPS on the GPx activity were found to differ in two respects, namely that LPS increased rather than decreased the activity of GPx in BALF cells; and that a posttreatment with taurine was more effective than a pretreatment in attenuating the change in GPx activity induced by LPS. From the results of two experiments with LPS alone, the GPx activity rose in the average by ∼3.1-fold relative to the control value (1.368 ± 0.083 U/min/ml vs. 0.445 ± 0.138 U/min/ml, respectively, P < 0.001, Fig. 16). Taurine was able to reduce this effect by about 60% when given for 3 consecutive days after a treatment with LPS (0.669 ± 0.290 U/min/ml vs. 1.368 ± 0.019, respectively, P < 0.05), and by 43% when given for the same period of time but before LPS (0.781 ± 0.177 vs. 1.369 ± 0.148, respectively, P < 0.05, Fig. 16). 4. Discussion

Fig. 9. Animals receiving taurine either before (A) or after (B) a treatment with LPS showed a significant reduction in the number of TUNEL-positive lung cells per HPF compared to animals treated with LPS alone. Each bar represents the mean ± S.E.M. for n = 6. *P < 0.001 vs. PBS + LPS and # P < 0.05 vs. LPS + PBS.

One of the earliest manifestations of ALI is the activation of free radical generation by the pulmonary endothelium and neutrophils, the upregulation of adhesion molecules, and the production of cytokines and chemokines promoting the recruitment of macrophages and neutrophils within the pulmonary microvasculature [35,36]. Following the migration of these phagocytic cells across the endothelium and epithelium into the alveolar space and their subsequent activation, they will release a variety of cytotoxic and proinflammatory factors, including proteolytic enzymes, ROS and NOS, cationic proteins, prostaglandins and additional cytokines [35]. The magnification of these events upon the recruitment of additional inflammatory cells and the continuous production of more cytotoxic mediators will ultimately overwhelm protective intracellular antioxidant mechanisms to cause airspace epithelial injury and respiratory failure as a result of oxidant–antioxidant imbalance [8,35]. The cellular damaging effects of ROS (including superoxide anion radical, hydrogen peroxide, hydroxyl radical, hypochlorous acid) and NOS (including nitric oxide and peroxynitrite) entails the lipid peroxidation of membrane phospholipids and culminates in the formation of MDA [8,37], the inactivation of antioxidant enzymes [8,38] and the oxidation of sulfhydryl-

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Fig. 10. Photomicrographs of sections from hamster lungs stained with ApoTag® Plus Peroxidase In Situ Apoptosis Detection kit. (A)–(C) and (D)–(F) show the results for animals receiving taurine as a pretreatment and as a posttreatment to LPS, respectively. Sections from animals receiving only PBS pH 7.4 showed no TUNEL positive apoptotic cells (A and D). Sections from animals serving as positive controls, treated with PBS pH 7.4 for 3 days before (B) or after (E) an i.t. treatment with LPS (0.02 mg), showed a modest increase in the number of TUNEL positive cells. The administration of taurine (50 mg/kg, i.p.) for 3 days before (C) or after (F) LPS reduced the number of TUNEL positive lung cells relative to samples from animals treated with LPS alone (magnification of 400×).

bearing molecules such as proteins [38] and GSH [37,39,40]. LPS along with oxidase-expressing epithelial and endothelial cells are considered to be major proinflammatory factors owing to their ability to prime and/or activate neutrophils to generate ROS. In the present study, the intratracheal instillation of bacterial LPS into the lungs of Golden Syrian hamsters led to an increase in the influx of leukocytes, in general, and of neutrophils, in particular, into the lungs, to an inflammatory response limited to the bronchial parenchyma, evidence of apoptotic death, lower GSH levels, and to BALF cells which, in comparison to those from control animals, exhibited enhanced expression of TNFR1, higher caspase 3 activity, with increased LPO, and altered activities of intracellular antioxidant enzymes. Although under basal conditions, alveolar epithelial lining fluid contains high levels of GSH, this molecule may be depleted upon its conversion to the oxidized (disulfide) form

Fig. 11. Animals receiving taurine either before (A) or after (B) a treatment with LPS showed a significant reduction in BALF caspase-3 activity compared to animals treated with LPS alone. Each bar represents the mean ± S.E.M. for n = 6. *P < 0.01 vs. PBS + LPS and # P < 0.05 vs. LPS + PBS.

GSSG during the removal of hydrogen peroxide and hydroperoxides by GPx, and because of the inability of lung cells to replenish it in the face of an inhibitory effect of oxidants and proinflammatory mediators on ␥-glutamylcysteine synthetase (␥GCS), the key enzyme for GSH synthesis [40]. There are several lines of evidence in support of an antioxidant role for taurine in vivo [23] and ex vivo [41] in spite of the existence of in vitro data suggesting that taurine is, at best, a weak antioxidant agent [42]. Indeed, experiments in various animal models of oxidative stress have verified that taurine of exogenous origin can increase the levels of GSH, a vital antioxidant for lung cells, and effectively limit the losses in GPx, SOD and CAT activities. Taurine could be preventing the loss of intracellular GSH by reducing the negative influence of oxidants and proinflammatory agents on redox-sensitive transcription factors regulating the gene expression

Fig. 12. Animals receiving taurine either before (A) or after (B) a treatment with LPS showed a significant reduction in BALF MDA formation compared to animals treated with LPS alone. Each bar represents the mean ± S.E.M. for n = 6. *P < 0.001 vs. PBS + LPS and # P < 0.001 vs. LPS + PBS.

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Fig. 13. The lung GSH content was significantly increased in animals receiving taurine before (A) LPS and was unchanged in animals receiving taurine after (B) LPS compared to animals treated with LPS alone. Each bar represents the mean ± S.E.M. for n = 6. *P < 0.001 vs. PBS + LPS.

Fig. 15. Animals receiving taurine either before (A) or after (B) a treatment with LPS showed a significant increase in BALF SOD activity compared to animals treated with LPS alone. Each bar represents the mean ± S.E.M. for n = 6. *P < 0.05 vs. PBS + LPS and # P < 0.05 vs. LPS + PBS.

of ␥GCS in the lungs [40]. Alternatively, taurine could be limiting LPO and, thereby, the formation of ROS causing the depletion of GSH [37,43]. This antioxidant action could also account for the protective properties that taurine manifests against free radical inhibition of antioxidant enzymes by LPS [44]. The antiinflammatory and injury-protecting actions of taurine appears to be dependent on its conversion to taurine chloramine (Tau-Cl) since only Tau-Cl, but not taurine, is found to inhibit cytokine production by LPS-stimulated human peripheral mononuclear cells [45] and murine BALF or peritoneal neutrophils [23,46]. Conceivably, the halide-dependent myeloperoxidase (MPO) of endotoxin-activated neutrophils will catalyze the peroxidation of chloride ions to generate HClO [23], which subsequently reacts with taurine to form Tau-Cl [23,48]. In addition to representing a mechanism for the removal of toxic HClO from the lungs, the formation of Tau-Cl will enable taurine to serve as a negative effector on the signaling pathway for the activation of nuclear transcription factors

associated with cytokine synthesis. In this regard, although it was earlier suggested that Tau-Cl produced its effects on the production of inflammatory mediators by interfering with the signaling pathway responsible for the activation of nuclear factor-␬B (NF-␬B) at a level upstream of I␬B kinase (IKK) activity [48], a later study has shown that part of the inhibitory effect of this taurine derivative is a more direct one and to favor the oxidation of Met45 of IKK-␣ [49]. In either case, the negative effect of Tau-Cl on the activity of the NF␬B signaling pathway may also regulate the production of MCP-1 and MCP-2, two chemokines involved in macrophage recruitment by activated neutrophils [47]. The effects of taurine on LPS-induced lung inflammation, apoptosis and oxidative stress were evaluated by administering this substance for 3 days by the intraperitoneal route both before and after LPS. In most instances, taurine was found to be more protective against LPS-induced alterations when administered before rather after LPS. Regardless of the order of administration of taurine relative to that of LPS, this sulfur-containing amino acid reduced

Fig. 14. Animals receiving taurine either before (A) or after (B) a treatment with LPS showed a significant increase in BALF CAT activity compared to animals treated with LPS alone. Each bar represents the mean ± S.E.M. for n = 6. *P < 0.001 vs. PBS + LPS and # P < 0.01 vs. LPS + PBS.

Fig. 16. Animals receiving taurine either before (A) or after (B) a treatment with LPS showed a significant reduction in BALF GPx activity compared to animals treated with LPS alone. Each bar represents the mean ± S.E.M. for n = 6. *P < 0.05 vs. PBS + LPS and # P < 0.05 vs. LPS + PBS.

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the influx of both total leukocytes and neutrophils into the lung airspaces, the ensuing intraalveolar inflammatory response, and the expression of TNFR1 on macrophages present in BAL following a challenge with LPS. Overall, these findings confirm the previously held view that taurine is a potent inhibitory modulator of the proinflammatory and immune response in the lung [50,51] and other major organs [52,53], possibly because of its attenuating action on the transcriptionally regulated production of proinflammatory and chemoattracting cytokines by alveolar macrophages [47,54] and other activated leukocytes [53,55] which, in turn, will promote the interaction of proinflammatory blood monocytes and neutrophils responsible for ALI, as well as their recruitment to the lungs [53]. In this situation, part of the effect taurine could be associated with an ability to prevent LPS-induced transendothelial migration of neutrophils into the lung by shortening their rolling velocity [56]. In addition, taurine may play a preventive role against LPS-induced ALI by reducing pulmonary artery pressure, hypoxia, pulmonary MPO activity and accompanying superoxide anion production, peripheral neutropenia, and the respiratory burst activity of neutrophils [27,28,53]. Further protection by taurine against ALI may stem from its ability to reduce the release of tumor necrosis factor-␣ from BALF cells such as neutrophils [23]. Considerable variation seems to exist among the results for the effects of LPS on the activity of antioxidants enzymes in BALF, lung homogenates or plasma reported by different laboratories. For example, while the present study finds that an intratracheal treatment of hamsters with LPS (0.2 ml of a 0.1 mg/ml solution) enhanced the activity of GPx and lowered that of CAT and SOD in BALF, a study conducted in rats found that within 6 h of an intraperitoneal treatment with LPS (500 ␮g/kg) the genes coding for antioxidant enzymes in the lungs were expressed in a divergent manner, with that for Mn-SOD being increased, that of CAT being decreased, and that of either Cu, Zn-SOD or GPx remaining unchanged [57]. Furthermore, it has been recently reported that the daily inhalation of a LPS solution by mice (0.5 mg/ml, for 5 days) led to lower CAT, GPx and SOD activities in plasma and BALF relative to control values [8]. Additional conflicting data also exists regarding the changes in lung SOD activity following a challenge with LPS. Thus, the intraperitoneal injection of LPS (20 mg/kg) to mice was found to induce a massive (>80%) decrease in extracellular SOD, the most abundant form of SOD in the lungs, but without affecting the cytoplasmic (Cu, Zn-SOD) and mitochondrial (Mn-SOD) SOD isoforms [58]. Although the exact reasons for the discrepancy in the antioxidant enzyme activities associated with the inflammatory response by LPS among laboratories awaits a systematic evaluation, they could stem from differences in the type of sample analyzed (plasma, BALF, tissue homogenate), endotoxin-related factors such as the duration of the exposure [8], the concentration of the solution administered [59], the route of administration [59], the dose used [59], and the animal species serving as the experimental subject [60]. In general, and irrespective of the trend of the changes in antioxidant enzyme activities that follows a challenge with LPS noted here and elsewhere, taurine was found to display a significant attenuating action against such changes. ALI by LPS is characterized by the generation of ROS which, if in excess, may be cause for the loss of cell function and cell death by apoptosis or necrosis [61]. LPS is known to initiate caspase activation and apoptotic death of neutrophils, macrophages and cells of alveolar walls within 24 h of its intratracheal instillation to mice, at least in part via CD14- and Fas-associated death domain-dependent mechanisms [62]. In the present study, LPS was found to double the number of cells showing apoptosis and to markedly raise the activity of caspase 3 activity of cells in BALF. In this context, it would appear that the apoptotic effect of LPS also extends to lung epithelial cells [26] and to other organs such as the brain [63] and liver [52] and, in all these instances, to involve stimulation of caspase activity

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and abrogation of intracellular calcium accumulation. The present results and those reported by other laboratories clearly indicate that taurine can protect against cell apoptosis by decreasing the expression or activation of caspases [12,63,64], the expression of apoptosis-related proteins [43], and the formation of injurious ROS [64]. In summary, the results of the present investigation verify that taurine can effectively protect the lung against cellular and biochemical alterations characterizing the ALI arising from an acute exposure to bacterial LPS. A greater protection by taurine is attained when this compound is administered before rather than after LPS. The benefits rendered by taurine may be related, at least in part, to its ability to limit the recruitment of proinflammatory leukocytes to the lungs. As a result, taurine will reduce the levels of phagocytic leukocyte-derived proinflammatory mediators, ROS and NOS responsible for oxidant–antioxidant imbalance, GSH depletion, LPO, apoptosis and acute cellular injury. On the basis of these results, a future study aimed at examining the role of taurine in lung inflammation due to a chronic exposure to LPS is warranted. Acknowledgement The authors would like to thank Forest Research Institute, a subsidiary of Forest Laboratories, Inc., Jersey City, New Jersey, for providing financial support to this study. References [1] Mei SHJ, McCarter SD, Deng Y, Parker CH, Liles CW, Stewart DJ. Prevention of LPSinduced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin 1. PLOS Med 2007;4:1525–37. [2] Jeyaseelan S, Chu HW, Young SK, Worthen GS. Transcriptional profiling of lipopolysaccharide-induced acute lung injury. Infect Immun 2004;72:7247–56. [3] Nagai A, Yasui S, Ozawa Y, Uno H, Konno K. Niacin attenuates acute lung injury induced by lipopolysaccharide in the hamster. Eur Respir J 1994;7:1125–30. [4] Johnston CJ, Finkelstein JN, Gelein R, Oberdörster G. Pulmonary cytokine and chemokine mRNA levels after inhalation of lipopolysaccharide in C57BL/6 mice. Toxicol Sci 1998;46:300–7. [5] Itoh T, Obata H, Murakami S, Hamada K, Kimura H, Nagaya N, Kangawa K. Adrenomedullin ameliorates lipopolysaccharide-induced acute lung injury in rats. Am J Physiol Lung Cell Mol Physiol 2007;293:L446–52. [6] Leiva M, Ruiz-Bravo A, Jimenez-Valera M. Effects of telithromycin in in vitro and in vivo models of lipopolysaccharide-induced airway inflammation. Chest 2008;134:20–9. [7] Nawrocka A, Papierz W, Bialasiewicz P, Stolarek R, Komos J, Nowak D. N-acetylcysteine and ambroxol inhibit endotoxin-induced phagocyte accumulation in rat lungs. Pulm Pharmacol Ther 1999;12:369–75. [8] Santos Valenca S, Silva Bezerra F, Aguiar Lopes A, Romana-Souza B, Marinho Cavalcante MC, Brando Lima A, et al. Oxidative stress in mouse plasma and lungs induced by cigarette smoke and lipopolysaccharide. Environ Res 2008;108:199–204. [9] Tschernig T, Janardhan KS, Pabst R, Singh B. Lipopolysaccharide-induced inflammation in the perivascular space in lungs. J Occup Med Toxicol 2008;30:3–17. [10] Liu SH, Xu XR, Ma K, Xu B. Protection of carbon monoxide inhalation on lipopolysaccharide-induced multiple organ injury in rats. Chin Med Sci J 2007;22:169–76. [11] Blackwell TS, Blackwell TR, Holden EP, Christman BW, Christman JW. In vivo antioxidant treatment suppresses nuclear factor-␬B activation and neutrophilic lung inflammation. J Immunol 1996;157:1630–7. [12] Brass DM, Hollingsworth JW, Cinque M, Li Z, Potyts E, Toloza E, et al. Chronic LPS inhalation causes emphysema-like changes in mouse lung that are associated with apoptosis. Am J Respir Cell Mol Biol 2008;39:584–90. [13] Uchiyama K, Takano H, Yanagisawa R, Inoue K, Naito Y, Yoshida N, et al. A novel water-soluble vitamin E derivative prevents acute lung injury by bacterial endotoxin. Clin Exp Pharmacol Physiol 2004;31:226–30. [14] Feng NH, Chu SJ, Wang D, Hsu K, Lin CH, Lin HI. Effects of various antioxidants on endotoxin-induced lung injury and gene expression: mRNA expressions of MnSOD, interleukin-1␤ and iNOS. Chin J Physiol 2004;47:111–20. [15] Gao J, Zeng B, Zhou L. The protective effects of early treatment with propofol on endotoxin-induced acute lung injury in rats. Middle East J Anesthesiol 2003;17:379–401. [16] Tumurkhuu G, Koide N, Dagvadorj J, Morikawa A, Hassan F, Islam S, et al. The mechanism of development of acute lung injury in lethal endotoxic shock using ␣-galactosylceramide sensitization. Clin Exp Immunol 2008;152:182–91. [17] Goraca A, Jósefowicz-Okonkwo G. Protective effects of early treatment with lipoic acid in LPS-induced lung injury in rats. J Physiol Pharmacol 2007;58:541–9.

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