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Vitamin A (VA) and its active metabolites play an essential role in lung airway function. Patients with moderate to severe chronic obstructive pulmonary disease (COPD) have a lower serum retinol concentration, and improvement of their 1-second Forced Expiratory Volume (FEV1) is achieved with VA supplementation. In order to test our hypothesis that the VA signaling pathway is compromised in COPD, we obtained 20 lung samples from COPD patients differing in the degree of emphysema as judged by their FEV% values. All were smokers or were exposed to secondhand smoke. Levels of VA (retinol/retinyl ester), tocopherols and carotenoids (lutein, beta-carotene) in these samples were determined using HPLC. Additional analytes beside VA were included for their known roles as antioxidants and modulators of VA-action. VA levels (retinol/retinyl ester) decreased significantly with the increase in severity of emphysema. Among other analytes, α-tocopherol levels fell by 25.8% in the severe emphysema group in comparison to the mild emphysema group, and lutein levels similarly decreased in severe compared to moderate emphysema groups. However, beta-carotene levels remained unchanged. Thus there is a significant linear correlation between lung VA-levels and the severity of emphysema. There was also a significant reduction in the levels of α-, δ-tocopherol and lutein in the severe emphysema group of COPD patients who either smoked or were exposed to smoke.

Consumption of α-lactalbumin as dietary protein offers a beneficial effect on breast cancer development. Breast cancer was developed by gavage administration of single dose of dimethylbenz(a)anthracene (DMBA) in female rats, maintained on AIN-76A diet with either 20% casein or α-lactalbumin (a component of whey protein). All tumors were detected by palpation. After approximately 130 days of DMBA administration, the animals were euthanized. There was a delay in the development of breast tumor in the α-lactalbumin group in comparison to the casein group. The number of tumors per rat was less in the α-lactalbumin group than that in the casein group at any time point up to 130 days after DMBA administration. Also the incidence of tumors and tumor volume was less in the α-lactalbumin group than those in the casein group. The casein group had a mixture of grade I, grade II and grade III tumors whereas the α-lactalbumin group had mostly grade I tumor. Furthermore, the proliferative index was significantly lower in the α-lactalbumin group than that in the casein group.

Intratracheal infusion of 2-chloroethyl ethyl sulfide (CEES), a mustard gas analog and a chemical warfare agent is known to cause massive damage to lung. The purpose of this study was to determine whether intratracheal CEES infusion causes neuronal damage. Histological, immunohistochemical, and Western blot studies indicated that CEES treatment caused dose-dependent increases in blood cell aggregation, microglial cell number, microglial activation, and brain inflammation. In addition, an increased expression of α-synuclein and a decreased expression of the dopamine transporter were observed. The results indicate that intratracheal CEES infusion is associated with changes in brain morphology mediated by an increase in α-synuclein expression, leading to neurotoxicity in a guinea pig model. These changes may be mediated by oxidative stress. Furthermore, the present study indicates for the first time that intratracheal infusion of a single dose of CEES can cause neuroinflammation, which may lead to neurological disorders in later part of life. © 2011 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:23–30 2012; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.20409

We reported earlier in a guinea pig model that exposure of 2-chloroethyl ethyl sulfide (CEES), a mustard gas analog, causes lung injury associated with the activation of tumor necrosis factor alpha (TNF-alpha), mitogen activated protein kinases (MAPK) signaling, and activator protein-1 (AP-1) transcription factor. Our earlier studies also revealed that antioxidant liposomes can be used as antidotes. Proinflammatory cytokines IL-1, IL-6, and TNF-alpha, either alone or in combination, can induce the activation of another group of transcription factors, namely SAF-1 (serum accelerator factor-1)/MAZ (Myc-associated zinc finger protein). Phosphorylation of SAF-1 via MAPK markedly increases its DNA-binding and transactivational potential. The objective of the present study was to investigate whether CEES exposure causes activation of IL-1 beta, IL-6, and SAF-1/MAZ and whether these effects can be prevented by antioxidant liposomes. A single dose (200 microL) of the antioxidant liposome mixture was administered intratracheally after 5 min of exposure of CEES (0.5 mg/kg). The animals were sacrificed either 1 h or 30 days after CEES exposure. CEES exposure caused an upregulation of proinflammatory cytokines IL-6 and IL-1 beta in the lung along with an increase in the activation of transcription factor SAF-1/MAZ. The antioxidant liposomes treatment significantly blocked the CEES-induced activation of IL-6, IL-1 beta, and SAF-1/MAZ. This might suggest that antioxidant liposomes might offer a potential therapeutic strategy against inflammatory diseases associated with activation of these bioactive molecules.

Recent studies suggest that calcium influx via L-type calcium channels is necessary for psychostimulant-induced behavioral sensitization. In addition, chronic amphetamine upregulates subtype Cav1.2-containing L-type calcium channels. In the present studies, we assessed the effect of calcium channel blockers (CCBs) on cocaine-induced behavioral sentitization and determined whether the functional activity of L-type calcium channels is altered after repeated cocaine administration. Rats were administered daily intraperitoneal injections of either flunarizine (40 mg/kg), diltiazem (40 mg/kg) or cocaine (20 mg/kg) and the combination of the CCB’s and cocaine for 30 days. Motor activities were monitored on Day 1, and every 6th day during the 30-day treatment period. Daily cocaine administration produced increased locomotor activity. Maximal augmentation of behavioral response to repeated cocaine administration was observed on Day 18. Flunarizine pretreatment abolished the augmented behavioral response to repeated cocaine administration while diltiazem was less effective. Measurement of tissue monoamine levels on Day 18 revealed cocaine-induced increases in DA and 5-HT in the nucleus accumbens. By contrast to behavioral response, diltiazem was more effective in attenuating increases in monoamine levels than flunarizine. Cocaine administration for 18 days produced increases in calcium-uptake in synaptosomes prepared from the nucleus accumbens and frontal cortex. Increases in calcium-uptake were abolished by flunarizine- and diltiazem-pretreatment. Taken together, the augmented cocaine-induced behavioral response on Day 18 may be due to increased calcium uptake in the nucleus accumbens leading to increased dopamine (DA) and serotonin (5-HT) release. Flunarizine and diltiazem attenuated the behavioral response by decreasing calcium uptake and decreasing neurochemical release.

It has been reported that there is an ameliorative effect of cigarette smoking on certain neurological responses and neurodegenerative disorders. The purpose of this study was to examine the neurochemical and neurobehavioral response of cigarette smoke (CS) in the adult male guinea pig brain. Both acute and chronic CS exposure enhanced locomotor behavior and caused a decrease in midbrain dopamine (DA) levels and corresponding increase in 3,4-dihydroxyphenylacetic acid (DOPAC) levels. In addition, CS caused a significant increase in the protein levels of the dopamine D1 and D2 receptors. CS caused a significant increase in the binding capacity of the D1 receptor and a significant decrease in the binding capacity of D2. Furthermore, CS caused a significant increase in the binding capacity of the dopamine transporter (DAT). The mechanism by which cigarette smoke exposure increases locomotor activity remains to be elucidated but may include modulation of dopamine neuron activity that emerges after repeated direct smoke exposure.

Exposure to mustard gas causes inflammatory lung diseases including acute respiratory distress syndrome (ARDS). A defect in the lung surfactant system has been implicated as a cause of ARDS. A major component of lung surfactant is dipalmitoyl phosphatidylcholine (DPPC) and the major pathway for its synthesis is the cytidine diphosphocholine (CDP-choline) pathway. It is not known whether the ARDS induced by mustard gas is mediated by its direct effects on some of the enzymes in the CDP-choline pathway. In the present study we investigated whether mustard gas exposure modulates the activity of cholinephosphotransferase (CPT) the terminal enzyme by CDP-choline pathway. Adult guinea pigs were intratracheally infused with single doses of 2-chloroethyl ethyl sulfide (CEES) (0.5 mg/kg b.wt. in ethanol). Control animals were injected with vehicles only. The animals were sacrificed at different time and the lungs were removed after perfusion with physiological saline. CPT activity increased steadily up to 4 h and then decreased at 6 h and stabilized at 7 days in both mitochondria and microsomes. To determine the dose-dependent effect of CEES on CPT activity we varied the doses of CEES (0.5-6.0 mg/kg b.wt.) and sacrificed the animals at 1 h and 4 h. CPT activity showed a dose-dependent increase of up to 2.0 mg/kg b.wt. of CEES in both mitochondria and microsomes then decreased at 4.0 mg/kg b.wt. For further studies we used a fixed single dose of CEES (2.0 mg/kg b.wt.) and fixed exposure time (7 days). Lung injury was determined by measuring the leakage of iodinated-bovine serum albumin into lung tissue and expressed as the permeability index. CEES exposure (2.0 mg/kg b.wt. for 7 days) caused a significant decrease of both CPT gene expression (approximately 1.7-fold) and activity (approximately 1.5-fold) in the lung. This decrease in CPT activity was not associated with any mutation of the CPT gene. Previously we reported that CEES infusion increased the production of ceramides which are known to modulate PC synthesis. To determine whether ceramides affect microsomal CPT activity the lung microsomal fraction was incubated with different concentrations of C(2)-ceramide prior to CPT assay. CPT activity decreased significantly with increasing dose and time. The present study indicates that CEES causes lung injury and significantly decreases CPT gene expression and activity. This decrease in CPT activity was not associated with any mutation of the CPT gene is probably mediated by accumulation of ceramides. CEES induced ceramide accumulation may thus play an important role in the development of ARDS by modulating CPT enzyme.

Mustard gas exposure causes adult respiratory distress syndrome associated with lung injury. The purpose of this study was to investigate whether an antioxidant, such as N-acetylcysteine (NAC), has any protective effect. Guinea pigs were given single exposure (0.5–6 mg/kg body weight) of 2-chloroethyl ethyl sulfide (CEES) as a mustard analogue intratracheally and maintained for various lengths of time (1 h to 21 days). Within 1 h of CEES infusion at 4 mg/kg, high levels of tumor necrosis factor α (TNF-α), ceramides, and nuclear factor κB accumulated in lung and alveolar macrophages. Both acid and neutral sphingomyelinases were activated within 4 h. These signal transduction events were associated with alteration in the oxygen defense system. Within 1 h of exposure to CEES (6 mg/kg body weight), there was 10-fold increase in the 125I-BSA leakage into lung tissue, indicating severe lung injury. Although low level of CEES exposure (0.5 mg/kg body weight) produced symptoms of chemical burn in lung as early as 1 h after exposure, the severity of edema, congestion, hemorrhage, and inflammation increased progressively with time (1 h to 21 days). Feeding of single dose of NAC (0.5 g) by gavage just before the CEES infusion was ineffective to counteract these effects. However, consumption of the antioxidant in drinking water for 3 or 30 days prior to CEES exposure significantly inhibited the induction of TNF-α, activation of neutral and acid sphingomyelinases, production of ceramides, activation of caspases, leakage of 125I-bovine serum albumin (125I-BSA) into lung tissue, and histological alterations in lung. Pretreatment with NAC for 3 and 30 days protected against 69–76% of the acute lung injury. Therefore, NAC may be an antidote for CEES-induced lung injury. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:177–184, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10076

Membrane phospholipids as well as fatty acid profile of cell membrane phospholipids are altered in tumorigenicity and malignancy. Synthesis of total cellular phosphatidylcholine (PC) can be used as a marker for membrane proliferation in neoplastic mammary gland tissues. Cholinephosphotransferase (CPT), the terminal enzyme in the de novo synthesis of PC, has an important role in regulating the acyl group of PC in mammalian cells. In this study, the effect of neoplasia on CPT was examined. The gene shows an elevated expression in cancerous (11-9-14) breast epithelial cell line when compared to that of normal non-tumorigenic (MCF-12A) breast epithelial cell line. Four nucleotide substitutions are observed in the cancer cell line. Of these, three are null mutations, but the third one shows an interesting serine to tyrosine substitution (at amino acid position 89 of our partial sequence which corresponds to position 323 of the CPT sequence reported as NM_020244 in GenBank) in 11-9-14 cells. The tyrosine is present in the right context of KSELYQDT, which directs tyrosine phosphorylation at the tyrosine site. Biochemical approach also reveals a 1.5-fold stimulation in CPT activity in 11-9-14 cells compared to that of the MCF-12A cells.

We have earlier identified the presence of a 36 kDa Ca2+-dependent phospholipid-binding protein (PLBP) in guinea pig alveolar type II cells. PLBP has been suggested to act as a mediator in facilitating and regulating intracellular surfactant assembly and delivery to the plasma membrane of type II cells for secretion into alveolar space. It has been reported that cigarette smoke exposure (CSE) causes a decrease in the surfactant activity in bronchial washings. We have also reported earlier that mainstream (MS) and sidestream (SS) CSE causes desensitization of β-adrenoreceptors in guinea pig alveolar type II cells. Since both Ca2+ and β-adrenoreceptors are involved in surfactant secretion and PLBP is involved in surfactant delivery, it is important to know whether CSE causes any change in the PLBP level in alveolar type II cells. In the present study, we have demonstrated that MS and SS CSE causes a significant increase in the levels of PLBP in alveolar type II cells (107 and 150%, respectively) and in lung lavage (42 and 125%, respectively) in comparison to that in sham control (430 ng/mg protein in alveolar type II cells and 780 ng/mg protein in lung lavage). The mechanism by which smoke exposure causes an elevation in the levels of PLBP in alveolar type II cells and lung lavage remains to be investigated.

In the present study, we generated antibodies in rabbits against two synthetic peptides, one based on peptide sequence from yeast CPT cDNA (position 86 to 98 of the amino acid sequence) and the other from our guinea pig CPT cDNA (it corresponds to amino acid positions 119 to 130 according to yeast CPT gene). The antibody titers were measured by both dot blot analysis and ELISA using Keyhole limpets hemocyanin coupled CPT peptides. The CPT antibody recognized a single band by Western blot analysis of proteins from guinea pig liver mitochondria and microsomes. The molecular weight of the protein recognized by Western blot analysis is close to the predicted molecular weight (46 kDa) of yeast CPT. Further analysis revealed that the antibody inhibited CPT activity in both subcellular fractions in a dose dependent manner, thus confirming the specificity of the antibody against both subcellular CPT.

The correlation of vitamin A with the binding characteristics of peripheral benzodiazepine receptors (PBRs) in testes have been implicated on the basis of findings of involvement of vitamin A in testicular physiology and the abundance of PBRs in testicular tissue. Both vitamin A and PBRs are involved in the control of cell proliferation and differentiation but no data exists regarding the relationship between them. In the present study, we have examined the effects of vitamin A deficiency on the affinity and density of PBRs in testes of guinea pigs. Weanling guinea pigs were divided into three groups: control, pair-fed control and vitamin A deficient. They were fed a complete semipurified diet. The vitamin A deficient diet was similar to the control diet except vitamin A palmitate was omitted. Vitamin A deficiency status was achieved after 90 days of feeding. Binding of [3H]Ro 5-4864, a specific ligand for peripheral benzodiazepine receptors was determined in whole homogenate of testicular tissue. There was a 77% decrease in the receptor density (Bmax) in vitamin A deficient group compared to control. The Bmaxvalues for control, pair-fed control and vitamin A deficient groups were: 12.4 ± 0.4, 8.8 ± 0.2 and 3.0 ± 0.6 pmol/g, respectively. The equilibrium dissociation constant (KD) values were also 86% decreased in the vitamin A deficient group compared to the other groups. The KD values for control, pair-fed control and vitamin A deficient groups were: 3.4 ± 0.7, 2.8 ± 0.5 and 0.5 ± 0.01, respectively. The decrease in the binding characteristics of PBRs in testes due to vitamin A deficiency was accompanied with a corresponding decrease in the levels of testosterone in plasma. These results suggest a close functional relationship of vitamin A with PBRs in testes.

Exposure of 2-chloroethyl ethyl sulfide (CEES) to guinea pigs causes lung injury by infiltration of neutrophils in interstitial lung spaces. A unique MAPK-regulated transcription factor, C/EBP (CCAAT-enhancer-binding protein), regulates the expression of intracellular adhesion molecule-1 (ICAM-1), involved in recruiting neutrophils in lung. The present study was to determine if CEES exposure causes activation of C/EBP, in particular the predominant β-isoform and if so whether it can be prevented by intratracheal delivery of an antioxidant liposome containing N-acetyl cysteine and tocopherols. Lung injury was developed in guinea pigs by intratracheal exposure of CEES (0.5 mg/kg). The antioxidant liposome was given intratracheally 5 min after CEES exposure, and the animals were sacrificed after 30 days. CEES exposure caused a 2.3-fold increase in the activation of C/EBP accompanied with a 45% and 121% increase in the protein level of C/EBP β and ICAM-1, respectively, and this effect was counteracted by the antioxidant liposome.

Toxic oil syndrome (TOS) is caused by ingestion of denatured edible oils. Even though the etiology and pathogenesis of this disease are not fully known, it is quite clear that generation of free radicals caused by ingestion of fatty acid anilides is responsible for the pathogenetic mechanism in many TOS patients. Fatty acid anilides may also alter the free radical status of lungs and erythrocytes; this possibility may shed some light on understanding toxic oil syndrome. The present study describes the effects of oral administration of fatty acid anilides on the activities of major enzymes involved in the oxygen defense systems of lungs and erythrocytes. Feeding fatty acid anilides caused an increase in the superoxide dismutase (SOD) activity in erythrocytes, whereas it caused a decrease in the SOD activity in lungs. GSH-Px activity was not significantly changed in erythrocytes but was decreased in lungs. Although the activity of catalase was increased only by a higher dose in the erythrocytes, it was not affected in the lung at any dosage. Even though the ingestion of fatty acid anilides caused an increase in the SOD activity in the erythrocytes and a decrease in the SOD activity in the lungs, there was an increase in the lipid peroxidation in both cases. The increase in lipid peroxidation in erythrocytes is probably caused by the accumulation of H2O2, and that in the lungs is due to the accumulation of superoxide anion.

beta-Adrenoreceptor has been studied in a clonal capillary endothelial cell line established from the vascular bed of the bovine adrenal medulla. [3H]Dihydroalprenolol ([3H]DHA) binding to the isolated plasma membranes from these cells has demonstrated the presence of beta-adrenoreceptors with two different affinities. The dissociation constants (Kd) have been found to be 0.27 +/- 0.09 x 10(-9) M and 2.96 +/- 0.31 x 10(-9) M, respectively with the corresponding Bmax of 5.1 +/- 0.05 and 70.0 +/- 0.2 pmol/mg protein, respectively. Inhibition of [3H]DHA binding to the beta-receptor by atenolol (a beta 1-antagonist) and ICI 118,551 (a beta 2-antagonist) has suggested that the IC50cor (= Ki) for atenolol and ICI 118,551 for high affinity site are 0.08 +/- 0.03 x 10(-12) M and 0.25 +/- 0.08 x 10(-12) M, respectively. This, therefore, indicates that both atenolol and ICI 118,551 are able to displace the bound ligand effectively but the beta 1-selective antagonist atenolol is 3 times more potent than its beta 2 counterpart, ICI 118,551. Displacement of [3H]DHA binding to the endothelial cell plasma membrane by the agonists isoproterenol, epinephrine and norepinephrine has established a relative order of Ki for these agents as isoproterenol (0.56 +/- 0.19 x 10(-9) M)epinephrine (0.77 +/- 0.26(-9) M)or = norepinephrine (0.71 +/- 0.24 x 10(-9) M) for the high affinity site. The corresponding values for the low affinity site, however, are 4.62 +/- 0.64 x 10(-9) M, 6.21 +/- 0.86 x 10(-9) M and 5.90 +/- 0.82 x 10(-9) M, respectively for the same agonists. Increased intracellular cAMP accompanied with cellular proliferation in the presence of isoproterenol has suggested not only the coupling of beta-adrenoreceptors to the adenylate cyclase system but also its involvement in endothelial cell proliferation.

We have studied the effects of short-term exposure of guinea pigs to cigarette smoke under both mainstream (MS) and sidestream (SS) conditions on the activities of major antioxidant enzymes and lipid peroxidation potential of erythrocytes. The smoke-exposed groups had an increase in the activity of superoxide dismutase (SOD), a decrease in the activities of glutathione peroxidase (GSH-Px) and NADPH generating enzymes, and no change in the activity of catalase. Furthermore, there was a significant increase in the in vitro lipid peroxidation potential of erythrocytes in both MS- and SS-exposed groups. However, the lipid peroxidation potential was higher in the MS-exposed group than that in the SS-exposed group.

It is known that thyroid hormone enhances the biosynthesis of phosphatidylcholine in the lung. The purpose of the present study was to investigate the effects of thyroid hormone on the activity of cholinephosphotransferase, the terminal enzyme in the CDP-choline pathway, in guinea pig lung mitochondria and microsomes. Intramuscular injection of triiodothyronine (T3, 0.25-4 mg/kg body wt) stimulated the activities of both mitochondrial and microsomal enzymes in a dose-dependent manner. However, the stimulation was much more pronounced in the microsomes than in the mitochondria. The stimulatory effect of T3 was blocked by the intraperitoneal injection of both actinomycin D and cycloheximide in the microsomes, whereas in the mitochondria, the hormonal effect was blocked only by cycloheximide. Thus, it is suggested that T3 stimulates not only the nucleocytoplasmic system for the de novo synthesis of the enzyme, but possibly also the regulation of the transport of the synthesized protein into the mitochondria. Furthermore, administration of T3 produced an increase in the uptake and incorporation of [14C]choline into phospholipids of lung slices in vitro. However, this effect was blocked by intraperitoneal injection of both actinomycin D and cycloheximide. Thus, the change in CPT activity by T3 in mitochondria is not reflected by enhanced incorporation of choline into phosphatidylcholine.

It is known that the composition of phospholipids in lung changes with age. The final step in the de novo synthesis of phosphatidylcholine, a major component of lung surfactant, by the CDP-choline pathway, requires the enzyme cholinephosphotransferase (CPT). Even though CPT has earlier been proposed to be located exclusively in the endoplasmic reticulum, we have recently demonstrated its presence also in the mitochondria. We have earlier reported a gestational variation of CPT activity in fetal mitochondria and microsomes. In the present study we examined the subcellular distribution of CPT activity in lung as a function of age. After birth, the microsomal CPT activity continued to increase until adulthood (24 wks of age), thereafter it gradually decreased. On the otherhand, the CPT activity of mitochondria continued to increase with the advancement of age and beyond 72 wks of age, it was approximately 2-fold higher than that of the microsomal fraction.

We report the validation of a reversed-phase gradient HPLC method allowing simultaneous quantification of retinol, retinyl esters, tocopherols and selected carotenoids in lung, liver and plasma of mouse, rat and guinea pig (gp) using a diode array detector. A significant species difference was observed regarding the distribution of retinol and retinyl esters. The levels of total retinol in lung, liver and plasma were in the following order: mouse >> rat > gp; rat >mouse > gp; and gp >> rat > mouse, respectively. Furthermore, comparison studies revealed similarities between the vitamin A profiles of human and gp lung samples.

The purpose of this study was to develop antioxidant liposomes as an antidote for mustard gas-induced lung injury in a guinea pig model. Five liposomes (LIP-1, LIP-2, LIP-3, LIP-4, and LIP-5) were tested with differing levels of phospholipid, cholesterol, phosphatidic acid, tocopherol (alpha, gamma, delta), N-acetylcysteine (NAC), and glutathione (GSH). A single dose (200 microL) of liposome was administered intratracheally 5 min or 1 h after exposure to 2-chloroethyl ethyl sulfide (CEES). The animals were sacrificed either 2 h after exposure (for lung injury study) or 30 days after exposure (for histology study). The liposomes offered 9%-76% protection against lung injury. The maximum protection was with LIP-2 (71.5% protection) and LIP-4 (75.4%) when administered 5 min after CEES exposure. Delaying the liposome administration 1 h after CEES exposure decreased the efficacy. Both liposomes contained 11 mM alpha-tocopherol, 11 mM gamma-tocopherol, and 75 mM NAC. However, LIP-2 contained additionally 5 mM delta-tocopherol. Overall, LIP-2 and LIP-4 offered significant protection by controlling the recruitment of neutrophils, eosinophils, and the accumulation of septal and perivascular fibrin and collagen. However, LIP-2 showed better protection than LIP-4 against the accumulation of red blood cells in the bronchi, alveolar space, arterioles and veins, and fibrin and collagen deposition in the alveolar space. The antifibrotic effect of the liposomes, particularly LIP-2, was further evident by a decreased level of lipid peroxidation and hydroxyproline in the lung. Thus, antioxidant liposomes containing both NAC and vitamin E are an effective antidote against CEES-induced lung injury.

2-Choloroethyl Ethyl Sulfide (CEES) exposure causes inflammatory lung diseases, including acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. This may be associated with oxidative stress, which has been implicated in the desensitization of beta-adrenergic receptors (β-ARs). The objective of this study was to investigate whether lung injury induced by intratracheal CEES exposure (2 mg/kg body weight) causes desensitization of β-ARs. The animals were sacrificed after 7 days and lungs were removed. Lung injury was established by measuring the leakage of iodinated-bovine serum albumin ([125I]-BSA) into lung tissue. Receptor-binding characteristics were determined by measuring the binding of [3H] dihydroalprenolol ([3H] DHA) (0.5–24 nM) to membrane fraction in the presence and absence of DLDL-propranolol (10 μ M). Both high- and low-affinity β-ARs were identified in the lung. Binding capacity was significantly higher in low-affinity site in both control and experimental groups. Although CEES exposure did not change KD and Bmax at the high-affinity site, it significantly decreased both KD and Bmax at low affinity sites. A 20% decrease in β2-AR mRNA level and a 60% decrease in membrane protein levels were observed in the experimental group. Furthermore, there was significantly less stimulation of adenylate cyclase activity by both cholera toxin and isoproterenol in the experimental group in comparison to the control group. Treatment of lungs with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase (PDE) could not abolish the difference between the control group and the experimental group on the stimulation of the adenylate cyclase activity. Thus, our study indicates that CEES-induced lung injury is associated with desensitization of β2-AR. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:59–70, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20265

Lutein, a dietary carotenoid, is a well known antioxidant. The major source of this carotenoid in humans is diet. We report here the presence of lutein, a dietary carotenoid in several guinea pig tissues (in decreasing order: liver > spleen > lung ≫ testis > kidney > plasma > eye but not in white adipose tissue). The presence of lutein in lung and other tissues may be significant in term of its antioxidant capacity of these organs.

Aim The present study aims to establish that cholinephosphotransferase (CPT), the terminal enzyme for the de novo biosynthesis of phosphatidylcholine (PC), can be used as a biomarker for breast cancer in an animal model. Main methods Breast cancer was induced by intragastric administration of dimethylbenz(a)anthracene (DMBA) in rats. The activity and expression of CPT were compared between normal breast tissues and breast tumors. To establish possible mechanistic model, we looked into other enzymes of PC biosynthesis as well as c-fos protein expression and DNA binding. Key findings CPT enzyme activity and its expression were significantly higher in breast cancer tissues relative to normal breast tissues. Corresponding to the increase in the CPT activity and its expression, c-fos activity and its expression were also increased in breast tumors. Significance The present study suggests that increased CPT activity and expression is associated with DMBA-induced breast cancer development.

The binding of [3H]Ro 5-4864 to peripheral benzodiazepine receptors (PBRs) was studied in normal and malignant submandibular glands of rats. The carcinoma was induced by implantation of 7,12-dimethylbenz[a]anthracene (DMBA) into the glands. [3H]Ro 5-4864 binding to normal and malignant submandibular glands indicated one population of binding sites with high affinity (KD of 3.4 and 4.4 nM for normal and malignant respectively) and saturability (Bmax) of 487 and 321 pmol/g tissue for normal and malignant respectively). Subcellular localization of PBRs indicates that mitochondria was the primary locale of the receptor in both cases and the decrease in Bmax was due primarily to a decrease in the binding capacity of PBRs in mitochondria.

Given the link between neurotoxicity and exposure to pollutants, the potential behavioral neurotoxicity of benzo(a)pyrene [B(a)P] was investigated. Studies have established that B(a)P requires metabolic activation to highly reactive species to elicit many of its adverse effects. This study investigated the perturbation of nervous system function by correlating behavioral changes with the metabolism of B(a)P, antioxidant enzyme levels and lipid peroxidation in selected brain regions. The neurobehavioral effects of single oral doses of B(a)P (25-200 mg kg(-1) body weight) on motor activity were examined in male F-344 rats at 2, 4, 6, 12, 24, 48, 72 and 96 h post treatment. Parent B(a)P and metabolites were measured at the above mentioned time points by reverse phase HPLC. The activity of several antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) and levels of malondialdehyde were determined at 6 and 96 h in both the striatum and hippocampus of B(a)P exposed rats. Suppression of motor activity (up to 70%) reached a maximum at 6 h, but was reversible at 96 h in all dose groups. The kinetics of disposition data show a strong link between B(a)P metabolism and the onset and duration of behavioral effects. Benzo(a)pyrene caused a 15-70% inhibition in the activity of superoxide dismutase and glutathione peroxidase and an enhancement in catalase and lipid peroxidation (up to 68%) in the striatum and hippocampus at 6 and 96 h post treatment, respectively. These findings suggest that B(a)P-induced acute neurobehavioral toxicity may occur through oxidative stress due to inhibition of the brain antioxidant scavenging system.

Mustard gas exposure causes inflammatory lung diseases. Many inflammatory lung diseases are associated with oxidative stress. Reactive oxygen species (ROS) are involved in the maintenance of physiological functions. In tissues, it is therefore essential to maintain a steady-state level of antioxidant activity to allow both for the physiological functions of ROS to proceed and at the same time preventing tissue damage. We have recently reported that mustard gas exposure decreases the overall activity of superoxide dismutase (SOD). In the present study, we investigated the effects of mustard gas on each of the three isozymes: SOD-1 (Cu/Zn), SOD-2 (Mn), and SOD-3 (extracellular). Adult guinea pigs were intratracheally injected single doses of 2-chloroethyl ethyl sulfide (CEES) (2 mg/kg body weight) in ethanol. Control animals were injected with vehicle in the same way. The animals were sacrificed after 7 days, and lungs were removed after perfusion with physiological saline. Lung injury was established by measuring the leakage of iodinated-BSA into lung tissue. Mustard gas exposure caused a significant increase in the activity of SOD-1 (35%). However, the SOD-3 activity which is the predominant type in lung was significantly decreased (62%), whereas no change was observed in SOD-2 activity. Thus the decrease in the total activity of SOD was primarily due to the SOD-3 isozyme. Northern blot analysis indicated 3.5-fold increased expression of SOD-1 in mustard gas exposed lung, but no significant change in the expression of SOD-2 and SOD-3 was observed. Mustard gas exposure did not cause mutation in the coding region of SOD-1 gene while causing modulation in expression levels. The protein levels of SOD-1, SOD-2, and SOD-3 were not altered significantly in the mustard gas exposed lung. Our results indicate that the overall decrease in the activity of SOD by mustard gas exposure is probably mediated by direct inactivation of the SOD-3 gene or the enzyme itself. This decrease in the activity of SOD-3 may be due to the cleavage of active form of the protein to an inactive form. The existence of active and inactive forms of SOD-3 as a result of shifts in Cys–Cys disulfide bonding has been described in human, recently. Studies are underway in our laboratory to investigate whether mustard gas induced inactivation of SOD-3 in lung is similarly mediated by a change in Cys–Cys disulfide bonding. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:142–149, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20128

Expression of peripheral benzodiazepine receptors (PBR) has been found in every tissue examined; however, it is most abundant in steroid-producing tissues. Although the primary function of PBR is the regulation of steroidogenesis, its existence in nonsteroidogenic tissues as well as in other cellular compartments including the nucleus suggests that there may be other roles for PBR. Our laboratory reported earlier a significant increase of PBR density in the nucleus of DMBA-induced malignant submandibular glands of rats, suggesting a role of PBR in nuclear events of peripheral tissues. Since then numerous studies have demonstrated the abundance of PBR in tumors. Numerous studies implicate a role for cholesterol in the mechanisms underlying cell proliferation and cancer progression. Based on studies with a battery of human breast cancer cell lines and several human tissue biopsies, Hardwick et al. suggested that PBR expression, nuclear localization, and PBR-mediated cholesterol transport into the nucleus are involved in human breast cancer cell proliferation and aggressive phenotype expression. The purpose of the present study is to confirm this hypothesis by developing an animal breast cancer model and correlating the above events with the breast cancer. Weanling rats were maintained on a diet containing animal protein (casein) for 30 days and then a single dose of DMBA in sesame oil (80 mg/kg) was administered by gavage to the animals. Control animals received the vehicle only. After 122 days of DMBA administration, the animals were sacrificed. All tumors were detected by palpation. B(max) of PBRs was 52.6% and 128.4% higher in the non-aggressive and aggressive cancer tissues, respectively, than that in normal tissues. Cholesterol uptake into isolated nuclei was found to be higher in both non-aggressive and aggressive tumor breast tissue than that in control tissue. There was also corresponding increase in B(max) of PBRs in the nucleus of cancer tissues. Furthermore, the nuclear nucleoside triphosphatase (NTPase) activity was found to be higher in aggressive tumor tissues than that in non-aggressive tumor tissues. In conclusion, these data suggest that PBR ligand binding, and PBR-mediated cholesterol transport into the nucleus may be involved in the development of mammary gland adenocarcinoma, thus participating in the advancement of the disease.

Sulfur mustard has been used as a vesicant chemical warfare agent. To understand the mechanism by which mustard gas exposure causes respiratory damage, we have used 2-chloroethyl ethyl sulfide (CEES) as a mustard analog. Our initial studies have shown that guinea pigs exposed to CEES intratracheally accumulate high levels of TNF-alpha. Accumulation of TNF-alpha leads to activation of both acid and neutral sphingomyelinases, resulting in high accumulation of ceramides, a second messenger involved in cell apoptosis. In addition, NF-kappa B was activated for a short period (1-2 h after exposure) as determined by mobility shift assay. Supershift assays indicated that both p50 and p65 of NF-kappa B were activated due to CEES exposure. However, NF-kappa B rapidly disappeared after 2 h. It is possible that the initial activation of NF-kappa B was an adaptive response to protect the cells from damage since NF-kappa B is known to inhibit TNF-alpha/ceramide-induced cell apoptosis. Since NF-kappa B disappeared after 2 h, the cells continued being damaged owing to accumulation of ceramides and activation of several caspases, leading to apoptosis.

The exact chemical composition of the red blood cell (RBC) membrane may vary depending on the methods used to isolate the membrane. We provide evidence here that RBC membrane can be fractionated by differential centrifugation and/or density gradient centrifugation into two distinct types, designated as ‘heavy membrane’ (HM) and ‘light membrane’ (LM). The amount of LM is twice that of HM on a per cell basis. HM and LM differ in some biochemical and electrophoretic properties. The total activities of Na+, K+- and Ca2+-ATPases, superoxide dismutase, glutathione peroxidase, catalase and glucose-6-phosphate and 6-phosphogluconate dehydrogenase are significantly higher in LM than HM on a per cell basis. While there is no significant difference in the specific activity of other enzymes between the two membranes, the specific activity of CA2+- ATPase is significantly higher in HM, whereas Na+, K+-ATPase activity is higher in LM. There is a remarkable difference in the distribution of major ghost polypeptides between these two membranes. Component I of spectrin, component III and a protein with mol. wt. of 165 KDa are present in smaller amounts, whereas component II of spectrin and proteins with mol. wt. of 145, 84 and 76 KDa, respectively, are present in higher amounts in HM than LM. Some proteins such as band 4.1,48 and 46 KDa are present only in LM, whereas some proteins with mol. wt. of 96,78 and 43 KDa, respectively are present only in HM. It has been confirmed that these two membranes are not representatives of either (a) right side-out vs. inside out vesicles, or (b) open vs. sealed membranes. Thus HM and LM are two distinct membrane fractions. It is suggested that some part of the membrane is denser than other parts, and during hemolysis of RBCs, the rbc membrane is spliced resulting in two populations, dense and light. ((Mol Cell Biochem 196: 141-149, 1999)

Both vitamin A and peripheral benzodiazepine receptors (PBRs) are involved in the control of cell proliferation and differentiation. The objective of this study was to determine whether vitamin A deficiency causes any modulation in the binding characteristics of the PBRs. Forty-five weanling guinea pigs were divided into three groups (control, pair-fed control, and vitamin A-deficient). Vitamin A-deficiency status was achieved after 90 days of feeding. It caused atelectasis, hyperplasia and metaplasia of alveolar epithelium, acute inflammation of the tracheobronchial tree, and a significant increase in the number of alveolar type II cells. In comparison to the control and pair-fed control groups, the lung of vitamin A-deficient animals had 40.6 and 42.8% less PBR density, respectively. There was no significant difference in the equilibrium dissociation constant (KD) of PBRs between the control and the pair-fed control groups, whereas the KD value was 77.7 and 60% higher in the vitamin A-deficient groups than in the control and the pair-fed control groups, respectively. Furthermore, vitamin A-deficiency caused a decrease in the binding capacity of PBRs in both nuclear and mitochondrial fractions. These results may suggest a close functional relationship between vitamin A and PBRs.

We have studied in guinea pigs the effects of cigarette smoke exposure on vitamin A (retinol) levels in plasma, lung, lung lavage, and liver. Smoke was generated from 1R3F cigarettes in a smoke exposure instrument designed by University of Kentucky Tobacco and Health Research Institute. Three-week-old male guinea pigs were exposed to mainstream, sidestream, or sham smoke, generated twice daily from three cigarettes for 6 weeks. In addition, some animals were kept as room controls for some time. After 6 weeks of smoke exposure, some animals were allowed to recover for 6 weeks without smoke. After 6 weeks of smoking, the plasma retinol levels were lower in both smoke exposed groups when compared to the values in the sham group. Furthermore, in comparison to the sham group, the mainstream and sidestream smoke exposed groups showed a 7.6- and 8.3-fold increase in the levels of lung retinol, respectively. After the 6-week recovery period, plasma retinol of both smoke-exposed groups reached the control levels. In contrast, withdrawal of smoking did not show such an effect on the lung retinol level in both mainstream or sidestream groups. Electronmicroscopy of the lungs showed deleterious alterations in the morphology of the lungs in both mainstream and sidestream groups. Although the mechanism(s) involved in the elevation of retinol content of the lung due to smoke exposure remains to be elucidated, it is of interest that elevation of retinol content and alteration of lung morphology occurred not only in the mainstream smoke exposed but also in the sidestream group.

Sicle cell trait subjects are potentially at risk withimicrovascualr compolications frequently associated with high altitude and extreme exertion. Studies conducted in our laboratory suggst that exposure of sickle cell trait subjects to treadmill exercise resulted in an increase not only in the susceptibility of red blood cells to oxidation but also in the density of RBC membrane. The trend of increased susceptibility of red cells to oxidation as a consequence of exercise was associated with relatively excessive production of H2O2, inefficient breakdown of H2O2 by GSH-Px- and catalase-catalyzed reactions, and inability/failure to show a transitory increase in the activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-generating enzymes.

Chlorpromazine, an antipsychotic drug, is found to inhibit Na+,K(+)-ATPase activity in rat brain microsomal membranes in vitro in concentration and time dependent manner but some inconsistency is observed when the effect was studied with respect to different temperatures. Various ligands and/or substrate affect the inhibition by chlorpromazine in different ways. The in vivo study with this drug shows that the activities of Na+,K(+)-ATPase, Ca2(+)-ATPase and acetylcholinesterase in the microsomal membranes of different organs are inhibit with increases in concentration or lengths of time of treatment and then levels off.

Rat testicular microsomal membrane fraction contains both Mg+2-dependent and Mg+2-independent Ca+2-ATPase activity. The latter activity is about two times higher than the former. Calcium ion required for maximum activation of Mg+2-independent Ca+2-ATPase in 3.0 mM, whereas for the dependent one it is 2.5 mM. Both the enzymes are resistant to cold shock upto seven days. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities, respectively. The pH optima for dependent one is 7.5, whereas for the independent one it is 8.5. Temperature optima for the former is 37 degrees C and for latter one it is 40 degrees C. Among all the nucleotides tested, ATP is found to be the best substrate for both the enzymes. The optimum concentration of ATP for dependent and independent enzyme activities are 3.0 mM and 1.5 mM respectively. Divalent metal ions like Zn+2, Ba+2 and Mn+2 have been found to inhibit Mg+2-dependent Ca+2-ATPase activity whereas Mg+2-independent Ca+2-ATPase activity is inhibited by the divalent ions except zinc which is found to stimulate the enzyme activity. Both the enzymes are inhibited by vanadate, EDTA and EGTA. I50, for vanadate is 0.05 and 0.125 mM for dependent and independent activities, respectively. Sulfhydryl groups modifying agents e.g., NEM, DTNB and chlorpromazine are found to affect the enzyme activities in different ways. Thus NEM and chlorpromazine are found to inhibit and DTNB stimulate the enzyme activities in both the cases.

Microsomal membranes isolated by sucrose density gradient centrifugation from mature toad ovary has been found to vary significantly in lipid composition and various enzyme activities in different seasons. Na+, K+—ATPase activity is the highest in breeding season (rainy season). Significantly the optimum temperature for enzyme activity is 30°C. The other enzyme Δ5-3β-hydroxysteroid dehydrogenase activity is also lower in hibernation period than other seasons. The total phospholipid, sterol and fatty acid contents differ significantly between seasons. The poly-unsaturated fatty acid, except arachidonic acid content in hibernation period is much lower than that during other seasons. The sterol content is also the lowest in this season. The present findings indicate that during hibernation period the membrane is more rigid and the metabolic activity of the animal is slow because of a lower level of various functionally important enzyme activities.

The effect of estradiol-17β and progesterone given separately as well as in combination on the rate of hydrogen peroxide formation and lipid peroxidation in the uteri of ovariectomized rats was studied. Estradiol in 3μg dose per day per animal elicited maximum stimulatory response and progesterone (100μg), on the other hand, was without any such effect. However, progesterone given along with estradiol completely prevented the effect due to the latter. In the same way, vitamin E, a well known antioxidant was found to be extremelv effective in protecting the uterus from the highly peroxidative action of estradiol-17β.