Co-encapsulation of gallium with gentamicin in liposomes enhances antimicrobial activity of gentamicin against Pseudomonas aeruginosa.

OBJECTIVES: The aim of this study was to enhance the antimicrobial efficacy of a liposomal gentamicin formulation with gallium metal (Lipo-Ga-GEN) against clinical isolates of Pseudomonas aeruginosa. METHODS: Sputum isolates of P. aeruginosa from cystic fibrosis patients were used to determine the MIC and MBC of Lipo-Ga-GEN. P. aeruginosa biofilms were formed and used to compare the minimum biofilm eradication concentration of the conventional drugs with that of Lipo-Ga-GEN. Quorum sensing (QS) molecule reduction of P. aeruginosa was determined by monitoring N-acyl homoserine lactone production using Agrobacterium tumefaciens reporter strain (A136). Viability of the cultured human lung epithelial cells (A549) was determined by Trypan Blue assay in order to assess Ga toxicity. RESULTS: MIC and MBC values indicated that gentamicin was more effective against a highly resistant strain of P. aeruginosa (PA-48913) when delivered as a Lipo-Ga-GEN formulation (256 mg/L free gentamicin versus 2 mg/L Lipo-Ga-GEN). Lipo-Ga-GEN was the only formulation that completely eradicated biofilms and blocked QS molecules at a very low concentration (0.94 mg/L gentamicin). The decrease in cell viability was less in A549 cells exposed to Lipo-Ga, suggesting that encapsulated Ga is safer. CONCLUSIONS: The results clearly indicate that the Lipo-Ga-GEN formulation is more effective than gentamicin alone in eradicating antibiotic-resistant P. aeruginosa isolates growing in a planktonic or biofilm community.

Biochemical mechanism of metallothionein--carbon tetrachloride interaction in vitro.

To elucidate the mechanism underlying the protective effect of metallothionein (MT) against carbon tetrachloride (CCl4) toxicity, in vitro experiments were carried out to study the interaction of metallothionein and CCl4. Results from this study showed that incubation of Cd,Zn-MT with CCl4 in the presence of hepatic microsomes and NADPH resulted in a time-dependent depletion of MT thiols with a concurrent reduction in the metal-binding sites of the protein. Moreover, this reaction also released Zn and Cd from MT. Results from experiments conducted to determine whether or not the CCl4-induced decrease in MT-thiol content was due to the scavenging of CCl4 metabolite(s) showed that the trichloromethyl radical, chloroform and phosgene as well as the products of CCl4-induced microsomal lipid peroxidation were not directly involved. Although covalent binding of 14CCl4 to MT was detected following incubation in the presence of a microsomal bioactivation system, it did not account for the CCl4-induced loss of MT thiol groups for the following reasons: (i) prior oxidation of sulfhydryl groups of MT by hydrogen peroxide did not alter the binding; and (ii) anaerobiosis did not alter the extent of covalent binding but obliterated the inhibitory effect of CCl4 on MT thiol content. Measurement of the thiol content of CCl4-treated MT after treatment with 1,4-dithiothreitol revealed that all the thiol groups that were lost subsequent to CCl4 treatment could be regenerated. These data suggest that CCl4-linked oxidation of MT, rather than the covalent binding of 14CCl4 metabolite(s), may be responsible for the CCl4-induced loss of metal binding sites of MT with the concurrent release of Zn and Cd. However, the precise role of the metal released during the oxidation of MT in CCl4 toxicity remains to be defined.

Alleviation of paraquat-induced lung injury by pretreatment with bifunctional liposomes containing alpha-tocopherol and glutathione.

Reactive oxygen species are known to play a key role in the development of acute lung injury, and such injury can be alleviated by pretreating the lung with a suitable antioxidant preparation. In this study, we evaluated and compared the antioxidant efficacy of two liposomal preparations: liposomes containing only alpha-tocopherol versus bifunctional liposomes containing both alpha-tocopherol and glutathione (GSH). alpha-Tocopherol liposomes (2 mg alpha-tocopherol/animal) or liposomes containing both alpha-tocopherol and GSH (2 mg alpha-tocopherol and 10 mumol GSH/animal) were intratracheally instilled into the lungs of rats 30 min prior to a challenge with paraquat dichloride (30 mg/kg, i.p.); animals were killed 24 hr post-paraquat challenge. Lungs of paraquat-challenged animals were damaged extensively as evidenced by increases in lung weight, indicative of edema, and decreases in lung activities of angiotensin converting enzyme (ACE) and alkaline phosphatase (AKP), indicative of endothelial and alveolar type II epithelial cell injuries, respectively. While the pretreatment of rats with alpha-tocopherol liposomes or liposomes containing both alpha-tocopherol and GSH significantly attenuated paraquat-induced changes in lung ACE activity to more or less the same extent, the bifunctional liposomal preparation conferred additional protection to alveolar type II epithelial cells, as evidenced by a significantly higher pulmonary AKP activity. Our results also showed that both liposomal preparations failed to ameliorate paraquat-induced lung edema despite a significant protection of pulmonary endothelial cells, suggesting that paraquat-induced edema formation may be independent of endothelial cell damage. In conclusion, liposome-associated antioxidants can protect the lung against an oxidant challenge, and the extent of protection appears to be related to the characteristics of each antioxidant formulation.

Nitrofurantoin-induced pulmonary toxicity. In vivo evidence for oxidative stress-mediated mechanisms.

The present study was carried out to examine whether nitrofurantoin-induced pulmonary toxicity in normal rats was mediated via oxidant stress mechanisms. The relative importance of the cellular antioxidant enzymes in nitrofurantoin toxicity was also assessed. For this, the pulmonary toxicity induced by nitrofurantoin in rats was evaluated at various time intervals after a single subcutaneous injection. Data from this study showed that nitrofurantoin (200 mg/kg, s.c.) resulted in transient but measurable lung damage as evidenced by the increases in wet lung weight/body weight ratio and decreases in lung angiotensin converting enzyme activity. A transient decrease in GSH concentrations with a concurrent increase in GSSG concentrations as well as an increase in lipid peroxidation levels (measured by the formation of diene conjugates and thiobarbituric acid reactants) were also evident in lungs of nitrofurantoin-treated rats. In addition, nitrofurantoin did not alter the pulmonary superoxide dismutase and glutathione peroxidase activities, but it did produce transient decreases in catalase and glutathione reductase activities. These data indicate that impairment of the ability of the lung to detoxify reactive oxygen species may play an important role in the development of nitrofurantoin-induced pulmonary toxicity. The results of the present study suggest that nitrofurantoin can damage the lungs of rats, probably through oxidative stress-mediated mechanisms. Also, our data have provided in vivo evidence for substantiating lipid peroxidation as a possible cause of lung damage.

Prophylaxis against lipopolysaccharide-induced lung injuries by liposome-entrapped dexamethasone in rats.

Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, stimulates phagocytes to generate metabolites that play an important role in the pathogenesis of acute lung injury. In this study, the prophylactic effect of liposome-entrapped dexamethasone (L-DEX) was evaluated in an animal acute lung injury model. Rats were pretreated intratracheally with L-DEX or dexamethasone phosphate (DEX) at a dose of 800 microg dexamethasone/kg body weight; 1 hr later, pretreated animals were challenged i.v. with LPS (Escherichia coli 0111:B4, 1 mg/kg body weight) and killed 24 hr later. Challenge of saline-pretreated animals with LPS resulted in lung injury, as evidenced by increases in wet lung weight and decreases in lung angiotensin-converting enzyme and alkaline phosphatase activities, injury markers of pulmonary capillary endothelial and alveolar type II epithelial cells, respectively. Also, LPS injection resulted in significant increases in plasma phospholipase A(2), thromboxane B(2), and leukotriene B(4) concentrations. The LPS challenge also increased pulmonary myeloperoxidase and elastase activities as well as chloramine concentrations, suggestive of neutrophil infiltration and activation of the inflammatory response. Pretreatment of animals with L-DEX was significantly more effective than pretreatment with the free drug in reducing lung inflammation and other lung injuries, as indicated by the appropriate injury markers used in this study. Our results suggested that the pulmonary delivery of liposome-entrapped anti-inflammatory drugs such as dexamethasone improves prophylactic efficacy in counteracting LPS-induced lung injury.

Protective effect of liposome-associated alpha-tocopherol against paraquat-induced acute lung toxicity.

The present study was undertaken to investigate whether alpha-tocopherol, entrapped in liposomes and delivered directly to the lung, could protect against paraquat-induced lung damage in the rat. Plain liposomes (composed of dipalmitoylphosphatidylcholine, DPPC) or DPPC/alpha-tocopherol liposomes were administered intratracheally to animals 24 hr prior to an intraperitoneal injection of paraquat (20 mg/kg); rats were killed 24 or 48 hr after paraquat treatment. Results of this study showed that lungs of animals treated with paraquat were damaged extensively as evidenced by an increase in lung weight and a significant reduction in lung angiotensin-converting enzyme (ACE) activity and cytochrome P450 concentration. Furthermore, paraquat treatment resulted in a significant decrease in reduced glutathione (GSH) concentrations and a marked elevation in microsomal lipid peroxidation levels as measured by the formation of diene conjugates. Pretreatment of rats with DPPC liposomes alone did not alter significantly the paraquat-induced changes of all parameters examined. On the other hand, pretreatment of rats with DPPC/alpha-tocopherol liposomes 24 hr prior to paraquat challenge resulted in a significant increase in pulmonary alpha-tocopherol concentrations and antagonized paraquat-induced changes in lipid peroxidation, GSH/GSSG ratio, lung ACE activity and cytochrome P450 concentrations. Results of this study suggested that alpha-tocopherol, delivered directly to the lung in a liposomal formulation 24 hr prior to paraquat administration, confers protection against paraquat-induced lung damage.

Treatment of LPS-induced tissue injury: role of liposomal antioxidants.

Tissue injury is a common occurrence in multiple organ failure, a possible clinical complication of Gram-negative bacterial sepsis. Gram-negative bacteria, in part through lipopolysaccharide (LPS), tumor necrosis factor, and other cytokines, activate neutrophils to increase oxygen consumption and produce reactive oxygen species (ROS). ROS have been suggested to play a critical role in the pathogenesis of multiple organ failure. Accordingly, we hypothesized that the susceptibility of tissues to ROS can be reduced by augmenting the antioxidant status of the affected tissues. Rats were challenged intravenously with LPS (Escherichia coli: 0111:B4) at a dose of 1 mg/kg body weight, and 0, 2, 4, or 6 h later were treated intravenously with plain liposomes or alpha-tocopherol liposomes (20 mg alpha-tocopherol/kg body weight); treated rats were then killed 24 h after LPS challenge. Animals challenged with LPS were extensively damaged in the liver, as evidenced by an increase in plasma alanine aminotransferase and aspartate aminotransferase activities, and also in the lung, as indicated by a decrease in pulmonary angiotensin-converting enzyme and alkaline phosphatase activities. The injection of LPS also resulted in increased myeloperoxidase activities in the two organs, suggestive of activation of the inflammatory response. Within the pulmonary and hepatic organs of LPS-challenged animals, the involvement of oxidative stress mechanisms was evident, because a significant decrease in reduced glutathione and an increase in lipid peroxidation were observed. In contrast, the administration of alpha-tocopherol liposomes in the post-LPS-challenge period resulted in a significant alleviation of both lung and liver injuries, evidenced by a general reversal of the altered biochemical indices toward normal among treated animals. The therapeutic effect was found to be greater when liposomal alpha-tocopherol treatment was given earlier during the development of injury. Plain liposomes administered immediately after LPS injection also protected hepatic and pulmonary tissues from injuries. However, unlike alpha-tocopherol liposomes, plain liposomes did not confer any beneficial effect when administered at later timepoints post-LPS injection. These data suggest that alpha-tocopherol, administered in a liposomal form, may serve as a potentially effective pharmacological agent in the treatment of LPS-induced tissue injuries.

N-acetyl cysteine attenuates acute lung injury in the rat.

The development of the adult respiratory distress syndrome (ARDS) in the critically ill patient is associated with a significant morbidity and mortality. The pulmonary dysfunction in ARDS is largely secondary to neutrophil-mediated oxidant injury. The purpose of these studies is to examine the effect of the antioxidant N-acetyl cysteine (NAC) on a rodent model of lung injury. We postulated that NAC might attenuate lung injury following intratracheal challenge with endotoxin (lipopolysaccharide; LPS). Male Sprague-Dawley rats were administered NAC systemically either before or after intratracheal administration of LPS. Lung injury was assessed by measuring the transpulmonary leakage of 125I-labeled albumin, pulmonary myeloperoxidase content, bronchoalveolar lavage fluid cell counts, pulmonary lipid peroxidation and histology. NAC administration significantly attenuated the LPS-induced increases in lung permeability (LPS: .24 +/- .08 vs. LPS + NAC: .12 +/- .03, p < .05) and reduced the LPS-dependent increase in lipid peroxidation. However, total and differential bronchoalveolar lavage cell counts and myeloperoxidase content were not affected by NAC pretreatment. Although neutrophil influx was unaffected, neutrophil activation as assessed by surface CD11b expression and chemiluminescence was significantly down-regulated by NAC. Importantly, NAC administration up to 2 h after endotoxin challenge was still able to significantly ameliorate LPS-induced lung injury. Our data suggests that the attenuation of acute lung injury by NAC in our rodent model is related to free radical scavenging and inhibition of the neutrophil oxidative burst, rather than by an effect on inflammatory cell migration. These results suggest novel approaches for therapeutic interventions in acute lung injury.

Liposomal antioxidants provide prolonged protection against acute respiratory distress syndrome.

BACKGROUND: We have previously shown that N-acetylcysteine (NAC), an antioxidant, in the resuscitation fluid after shock prevents lung injury in response to lipopolysaccharide (LPS) by inhibiting chemokine generation by alveolar macrophages in the lung. However, the protection was short-lived. We hypothesized that liposomal (Lip) NAC delivered intratracheally might be delivered directly to the target cells and exert prolonged effect. METHODS: Sprague-Dawley rats were bled to a blood pressure of 40 mm Hg for 1 hour and resuscitated with shed blood and equal volume of Ringer's lactate. In some studies 500 mg/kg NAC was included in the resuscitation fluid. Thirty minutes later, 150 microl LipNAC (9.4 mg/kg NAC) was given intratracheally. One hour and 18 hours after resuscitation, LPS (30 microg/kg) or saline was given intratracheally. Lung injury was assessed by permeability to (125)I-albumin, bronchoalveolar lavage neutrophils and lung myeloperoxidase. The cytokine-induced neutrophil chemoattractant (CINC) expression in the lung was assessed by Northern blot. RESULTS: At the early time point, both NAC and LipNAC protected the lung with the effects in significantly reducing the increases in transpulmonary albumin flux, neutrophil influx and myeloperoxidase in the lungs of shock/LPS rats. However, by the late time point, only LipNAC retained its salutary effect. This correlated well with persistent ability to prevent CINC increase. In addition, Lipalpha-tocopherol (alpha-T) and LipNAC/alpha-T were tested and determined to be effective to protect the lung. CONCLUSIONS: Liposomal encapsulation of antioxidants at low dose provides long lasting protection against acute respiratory distress syndrome after shock. This may represent a novel treatment approach.

The pulmonary uptake of intravenously administered liposomal alpha-tocopherol is augmented in acute lung injury.

The present study was carried out to investigate whether the intravenous administration of liposomal alpha-tocopherol can result in a significant localization of the antioxidant in the injured lung. Male Sprague-Dawley rats were injected with paraquat dichloride (20 mg/kg, ip.) and 4, 24 or 48 h later, they were given an intravenous injection of a liposomal alpha-tocopherol preparation (20 mg alpha-tocopherol in 128 mumoles liposomal lipid/kg) labelled with [14C]dipalmitoylphosphatidylcholine (DPPC) and [3H]alpha-tocopherol. Animals were killed and their lungs removed for analysis 24 h after liposomal treatment. To demonstrate whether the extent of uptake of radioactive alpha-tocopherol liposomes was directly related to the extent of residual lung injury, additional groups of animals were also injected with higher doses (30 and 40 mg/kg body weight) of paraquat dichloride and 48 h later, were treated with liposomal alpha-tocopherol; animals were then killed 24 h after liposomal alpha-tocopherol treatment. The intraperitoneal injection of paraquat dichloride resulted in time- and dose-dependent decreases in angiotensin converting enzyme and alkaline phosphatase activities suggesting that the toxicant injures both the capillary endothelial cells and alveolar type II epithelial cells, respectively. The recovery of intravenously administered radioactive alpha-tocopherol in the lungs of saline-treated animals was found to be about 2% of the initial dose 24 h post-liposomal treatment. However, in paraquat-treated animals, there was an increased localization of the labelled alpha-tocopherol to the lung, resulting in a difference of pulmonary delivery by as much as 2-3 fold compared to that in a normal lung. The 3H/14C ratio, representing the recovery of [3H]alpha-tocopherol and [14C]liposomes, was practically constant and there was a linear relationship between the measurable lung injury index and the corresponding recovery of radiolabelled alpha-tocopherol in the lung. Our results appear to suggest that the residual pulmonary injury augments the delivery of liposomal alpha-tocopherol to the lung.

Liposomes promote pulmonary glucocorticoid delivery.

The beneficial effects of glucocorticoids in treating pulmonary inflammatory disorders are complicated by systemic adverse effects. Thus, a possible reduction in dosage and dosing frequency would be advantageous, particularly for patients requiring high doses of the drug. We believe that this can be achieved by developing formulations that increase the retention of glucocorticoids in the lung and a liposome-based drug delivery system may be useful. In the present study, we examined the pulmonary delivery of a liposomal glucocorticoid formulation. Male adult rats were intratracheally instilled with free [3H]dexamethasone (DEX) or [14C]liposome-entrapped [3H]dexamethasone (L-DEX) (800 microg DEX kg body weight) and animals were killed at different times within a 72-h treatment period. Pulmonary retention of [3H]DEX in animals instilled with free DEX was found to be approximately 1.5% of the administered dose 4 h post-instillation, with no radioactivity detectable 24 h post-instillation. Liposome encapsulation of the drug altered the pulmonary retention of DEX with about 34% and 8% of radioactivity remaining in the lung at 4 and 24h post-instillation, respectively. The intratracheal instillation of free DEX or L-DEX reduced the number of leukocytes in peripheral blood to a similar extent (50% of control values) at 4h. However, unlike free-DEX-treated animals whose leukocyte counts returned to control levels by 24h, the circulating leukocyte counts of L-DEX-treated animals remained depressed in the same period. Furthermore, DEX-induced changes in ACTH levels were less evident in animals treated with the liposomal formulation than those treated with free DEX. Our data suggest that the administration of liposome-entrapped DEX has the distinct advantage of enhancing the anti-inflammatory activity of the drug and therefore, possibly reducing its need for frequent administration.

Prevention of phorbol myristate acetate-induced acute lung injury by alpha-tocopherol liposomes.

Phorbol-myristate acetate (PMA) is commonly used to produce experimental edema and other tissue injuries in the lung. Lung injuries induced by the administration of PMA has been shown to be mediated mainly by neutrophils. Neutrophils recruited to the lower respiratory tract may damage lung tissues by releasing reactive oxygen species, neutral proteases, and lysosomal enzymes. The present study was conducted to investigate whether alpha-tocopherol, entrapped in dipalmitoylphosphatidylcholine liposomes and delivered directly to the lung, could counteract some of the PMA-induced lung injuries. Plain liposomes or alpha-tocopherol containing liposomes (8 mg alpha-tocopherol/kg body weight) were intratracheally instilled into the lungs of rats 24 hr prior to PMA exposure (25 micrograms/kg) and treated rats were killed 3 hr later. Lungs of control animals exposed to PMA developed an increase in lung weight and lipid peroxidation as well as a decrease in lung angiotensin converting enzyme (ACE) and alkaline phosphatase (AKP) activities. PMA treatment also caused an increase in myeloperoxidase (MPO) activity in the lung, suggestive of neutrophil infiltration. Pretreatment of PMA-treated rats with plain liposomes had no effect on PMA-induced injuries. In contrast, pretreatment of rats with liposomal alpha-tocopherol, 24 hr prior to PMA administration, resulted in a significant elevation of pulmonary alpha-tocopherol concentration, accompanied by a concomitant reduction in MPO activity and reversal of PMA-induced changes in lung edema, lipid peroxidation, ACE and AKP activities. These results appear to demonstrate that the intratracheal administration of a liposome-associated lipophilic antioxidant, such as alpha-tocopherol, can significantly ameliorate the toxic effects of reactive oxygen species, putatively released from PMA-stimulated pulmonary target cells and infiltrating neutrophils.

Liposomal alpha-tocopherol alleviates the progression of paraquat-induced lung damage.

The present study was carried out to investigate the efficacy of liposome-associated alpha-tocopherol in treating pulmonary damage caused by paraquat exposure. alpha-Tocopherol liposomes (8 mg alpha-tocopherol/kg body weight) or plain liposomes were intratracheally instilled into the lungs of rats 24 h after paraquat treatment (20 mg/kg, ip); treated animals were killed 8, 24 or 48 h after administration of the liposomal preparations. Lungs of animals exposed to paraquat were extensively damaged as evidenced by an increase in lung weight and decreases in pulmonary angiotensin converting enzyme and alkaline phosphatase activities. Also, paraquat treatment resulted in a significant reduction in glutathione (GSH) concentration in the lung and an elevation in microsomal lipid peroxidation levels, as measured by the formation of diene conjugates. Treatment of paraquat-injected rats with plain liposomes did not significantly alter paraquat-induced changes of all parameters examined. On the other hand, treatment of rats with alpha-tocopherol liposomes, 24 h after paraquat administration, resulted in a significant increase in pulmonary alpha-tocopherol concentrations as well as a reduction in paraquat-induced changes in lipid peroxidation, GSH concentration, and lung angiotensin converting enzyme and alkaline phosphatase activities. The results of the present study suggest that alpha-tocopherol, administered directly to the lung in a liposomal form, may serve as a potentially effective pharmacological agent in the treatment of paraquat-induced lung injury.

Liposomes in pulmonary applications: physicochemical considerations, pulmonary distribution and antioxidant delivery.

The application of liposomes for improved drug delivery to the lung is promising. Liposome-mediated pulmonary drug delivery promotes an increase in drug retention-time in the lung and more importantly, a reduction in extrapulmonary side-effects, invariably resulting in enhanced therapeutic efficacies. The engineering of an effective liposomal drug formulation for inhalation therapy must take into consideration the leakage problem associated with the nebulization process; vesicle stability and release kinetics within the pulmonary milieu; and, the altered pharmacokinetics of the entrapped drug. The delivery of liposome-entrapped antioxidants via the tracheobronchial route has been found to be very useful in increasing the half-times of the administered agents, thus providing a sustained release effect for prolonged drug action. The entrapment in liposomes of alpha-tocopherol, an extremely insoluble but highly effective antioxidant, has been shown to be very effective in ameliorating oxidant-induced injuries in the lung. The use of bifunctional liposomes containing two antioxidants have been determined to provide excellent resistance to an oxidative challenge and appears to hold promise for improved clinical applications in antioxidant therapy.

Incorporation of alpha-tocopherol in liposomes promotes the retention of liposome-encapsulated glutathione in the rat lung.

The present study was undertaken to investigate whether alpha-tocopherol incorporated in liposomes could improve the retention of entrapped glutathione (GSH) in the lung following intratracheal instillation in rats. Rats were treated with a single dose of [3H]GSH entrapped in liposomes with or without 30 mol% alpha-tocopherol and killed 0, 24 or 48 h later. The retention of GSH in the lung was assessed by measuring the recovery of either 3H-label or GSH in the lung. Animals instilled with free [3H]GSH were found to retain only 2% of the administered dose at 24 h after treatment and no detectable radioactivity at 48 h. Liposome encapsulation altered the pulmonary retention of GSH with 18 and 10% of radioactivity remaining in the lung at 24 and 48 h post-treatment, respectively. The instillation of GSH encapsulated in alpha-tocopherol-containing liposomes resulted in the highest level of GSH retention in the lung, namely 37 and 30% of the administered GSH dose at 24 and 48 h, respectively. Results from Sepharose 4B column chromatography revealed that lung homogenates, obtained from rats instilled with GSH entrapped in alpha-tocopherol-containing liposomes, 24 and 48 h earlier, contained 2 eluted GSH-related components--one associated with the liposomal lipid marker in the void volume and the other as free GSH tripeptide, suggesting a slow sustained release effect mediated by the liposomal formulation. The same liposome preparation containing both alpha-tocopherol and GSH also conferred better protection against FeADP-induced lipid peroxidation than liposomes containing either alpha-tocopherol or GSH alone, indicative of a potentially effective antioxidant formulation for treating oxidative lung injury.

Pulmonary uptake of liposome-associated alpha-tocopherol following intratracheal instillation in rats.

This study examined the uptake and subcellular distribution of alpha-tocopherol in the lung following intratracheal instillation of liposome-associated alpha-tocopherol in rats. The liposomal suspension was composed of dipalmitoylphosphatidylcholine (DPPC) and alpha-tocopherol (molar ratio 7:3), labelled with [3H]alpha-tocopherol and [14C]cholesterol. Following intratracheal administration of the liposomal preparation (2 mg alpha-tocopherol/animal), the recovery of [3H]alpha-tocopherol in the lung was maximal (87% of initial dose) 1 h after treatment; thereafter, alpha-tocopherol levels remained relatively high (no less than 73% of initial dose) for the rest of the 72-h experimental period. This treatment effect/resulted in a 16-fold increase in pulmonary total alpha-tocopherol concentration 72 h post-instillation. No radioactivity was detected in the blood, liver, kidney, pancreas, spleen and heart of animals during the 72-h experimental period. [3H]alpha-Tocopherol was recovered largely from cytosolic (45%) and nuclear (36%) fractions of lung and to a lesser extent, from microsomal (11%) and mitochondrial (9%) fractions. Chromatographic analysis of the subcellular fractions revealed that [3H]alpha-tocopherol was co-eluted with 14C-labelled liposomal lipids. Our in-vitro study, involving the incubation of Fe(3+)-ADP (a pro-oxidant) with mitochondrial or microsomal fractions isolated from lung tissues of animals treated with liposome-associated alpha-tocopherol, provided evidence that alpha-tocopherol levels present in the membranes of these subcellular fractions were sufficient to protect against oxidant-induced lipid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Protective effect of liposomal alpha-tocopherol against bleomycin-induced lung injury.

The clinical use of bleomycin in the treatment of squamous cell carcinomas, lymphomas and testicular tumours has been limited by its toxic effects, the most serious being pulmonary injury. The present study was undertaken to investigate whether alpha-tocopherol, incorporated in liposomes and delivered directly to the lung, could offer protection against bleomycin-induced lung damage and fibrosis in the rat. Animals were administered, intratracheally, plain liposomes (composed of dipalmitoylphosphatidylcholine, DPPC) or alpha-tocopherol-containing liposomes (8 mg alpha-tocopherol/kg body weight) and 30 min later, were exposed to bleomycin sulphate (4 units/kg body weight) by intratracheal instillation; treated animals were killed 21 days later. Results of this study showed that lungs of animals treated with bleomycin were seriously damaged, as evidenced by significant histological changes and increases in lung weight, lipid peroxidation, myeloperoxidase activity and hydroxyproline content as well as decreases in lung angiotensin converting enzyme (ACE) and alkaline phosphatase (AKP) activities. Pretreatment of rats with plain liposomes alone did not alter significantly the bleomycin-induced changes of all parameters examined. In contrast, pretreatment of rats with alpha-tocopherol liposomes 30 min prior to bleomycin administration resulted in little or no histological changes and conferred a significant protection against bleomycin-induced changes in edema, lipid peroxidation, hydroxyproline content, and ACE, AKP and myeloperoxidase activities. Results of this study suggested that pretreatment of rats with alpha-tocopherol liposomes can provide a significant protection against bleomycin-induced lung injury.

Intratracheally administered liposomal alpha-tocopherol protects the lung against long-term toxic effects of paraquat.

Paraquat is a broad-spectrum herbicide known to produce lung injury via oxidative stress-mediated mechanisms. Different pharmacological strategies have been explored to reduce the formation of these reactive oxygen species and/or prevent their toxic effects in the treatment of paraquat poisoning. The present study was carried out to investigate whether the antioxidant alpha-tocopherol, incorporated into liposomes and delivered directly to the lungs of rats, could protect the organ against the long-term toxic effects of paraquat. Plain liposomes (composed of dipalmitoylphosphatidylcholine, DPPC) or alpha-tocopherol liposomes (8 mg alpha-tocopherol/kg body weight) were administered intratracheally to animals 24 h prior to an intraperitoneal injection of paraquat dichloride (20 mg/kg) and rats were killed 0, 1, 4, 6, 8, 10, 12, 16, 19 or 24 days after paraquat treatment. Results of this study showed that lungs of animals treated with paraquat were extensively damaged, as evidenced by significant increases in lung weight and decreases in lung angiotensin converting enzyme (ACE) and alkaline phosphatase enzyme (AKP) activities. Moreover, paraquat treatment: resulted in a significant reduction in the number of neutrophils in the blood of rats with a concurrent increase in the pulmonary myeloperoxidase activity, suggestive of neutrophil infiltration in the lungs of treated animals. Pretreatment of rats with liposomes alone did not significantly alter the paraquat-induced changes of all parameters examined. On the other hand, pretreatment of rats with alpha-tocopherol liposomes, 24 h prior to paraquat challenge, attenuated paraquat-induced changes in ACE, AKP and myeloperoxidase activities but failed to prevent increases in lung weight. Thus, pretreatment of rats with liposome-associated alpha-tocopherol appears to protect the lung against some of the toxic effects of paraquat.

Age-related differences in iron-nitrilotriacetate hepatotoxicity in the guinea pig: role of copper metallothionein.

This study was concerned with the role of Cu and Cu-MT (metallothionein) in oxidative stress. Because hepatic Cu and Cu-MT concentrations are known to be high in the 3-day-old guinea pigs but decline to low adult levels by 7 days of life, the hepatotoxicity of ferric nitrilotriacetate (FeNTA) in the developing guinea pig was used as the experimental model in the present study. Results of this study showed that the hepatotoxic response to FeNTA (3.5 mg Fe /kg i.p.) as measured by elevation in serum aspartate aminotransferase activity, increase in lipid peroxidation, decrease in reduced glutathione/oxidized glutathione ratio and histopathological changes was higher in 3-day-old than in 7-day-old and adult guinea pigs. Furthermore, pretreatment of 7-day-old guinea pigs with cupric sulfate (0.5 mg Cu++/kg i.p.) increased hepatic Cu and Cu-MT levels and enhanced susceptibility to FeNTA. FeNTA treatment resulted in the oxidation of MT thiolates and reduction in the metal binding capacity and Cu content of MT in the 3-day-old and Cu-pretreated 7-day-old animals, providing evidence for the interaction between Cu-MT and cellular oxidants. In vitro study with FeNTA and hepatic microsomes revealed no age-related differences in microsomal lipid peroxidation; however, this parameter was stimulated in the presence of control or heat-treated cytosols isolated from 3-day-old but not those of 7-day-old animals. These observations were consistent with the involvement of Cu-MT, a heat-stable metalloprotein, in the sensitization of hepatic tissues to oxidative injury in the 3-day-old animal. Moreover, in vitro study involving the use of D-penicillamine, a Cu chelating agent, showed that the sensitization effect of Cu-MT was mediated by Cu ions. The results of this study suggest that Cu-MT may have a prooxidative property and tissues with high Cu-MT levels may be particularly susceptible to oxidative stress.

Prophylaxis against lipopolysaccharide-induced acute lung injury by alpha-tocopherol liposomes.

OBJECTIVE: To investigate whether intravenously administered liposomal alpha-tocopherol can protect the lung from the injurious action of Escherichia coli lipopolysaccharide (LPS). DESIGN: Prospective, randomized animal study. SETTING: Government research laboratory. SUBJECTS: Twenty adult male Sprague-Dawley rats. INTERVENTIONS: Animals were intravenously pretreated with alpha-tocopherol liposomes (20 mg alpha-tocopherol/kg body weight), plain liposomes, or saline. Twenty-four hours later, pretreated animals were challenged with an intravenous injection of LPS (E. coli 0111:B4, 1 mg/kg body weight), and killed 2 hrs after LPS challenge. MEASUREMENTS AND MAIN RESULTS: Challenge of saline-pretreated animals with LPS resulted in lung injuries as evidenced by an increase in wet lung weight and a reduction in pulmonary angiotensin converting enzyme (25%) and alkaline phosphatase (28%), injury markers of lung endothelial and epithelial type II cells, respectively. Also, LPS administration resulted in an increase in pulmonary myeloperoxidase and protease activities, indicative of a neutrophilic inflammatory response. Pretreatment of animals with liposomal alpha-tocopherol significantly attenuated the LPS-induced edematous lung weight response, and reduced the extent of injuries to the pulmonary endothelial and epithelial cells, demonstrated by a significantly smaller reduction in the corresponding enzyme marker activities. CONCLUSION: These results suggest that augmentation of the pulmonary antioxidant status can ameliorate LPS-induced lung injuries mediated by oxidative stress mechanisms.