Timothy W. Robison

Transfection with γ-glutamyl transpeptidase enhances recovery from glutathione depletion using extracellular glutathione

Glutathione (L-gamma-glutamyl-L-cysteinylglycine) is an important constituent of the antioxidant and detoxifying mechanisms of cells. The plasma membrane bound enzyme, gamma-glutamyl transpeptidase (GGT), catalyzes the first step in the degradation of extracellular glutathione, the components of which are then used for de novo glutathione synthesis. We tested the hypothesis that an increase in GGT activity would enhance the utilization of extracellular glutathione by cells challenged with a glutathione-depleting agent. A eukaryotic system stably overexpressing GGT (nearly 200-fold) was developed by transfection of NIH-3T3 fibroblasts with a human placental GGT cDNA. These cells and controls were incubated for 30 min with 1 mM diethyl maleate, which caused approximately 80% intracellular glutathione depletion. Glutathione was added to the medium and cells were allowed to resynthesize intracellular glutathione. The transfected cells used extracellular glutathione much more efficiently than controls in terms of both the concentration dependence and the rate of glutathione resynthesis. Serine-borate, a competitive inhibitor of GGT, blocked the restoration of intracellular glutathione. The results support the hypothesis that the increase in GGT activity that occurs in some toxicologic or pathologic conditions could provide protection against glutathione depletion.

Development and characterization of rabbit tracheal epithelial cell monolayer models for drug transport studies

AbstractPurpose. The objective of this study was to investigate how culture conditions would affect the morphological, functional, and permeability characteristics of rabbit tracheal epithelial cell layers being considered for drug transport studies. Methods. Rabbit tracheocytes were isolated by protease treatment and plated onto collagen-treated permeable supports at 1.3 × 106 cells/cm2. After 24 hr, cell layers were cultured either air-interfaced (AIC) on their apical surface or under conventional liquid covered conditions (LCC). Results. Scanning electron microscopy revealed mature cilia in AIC cell layers and ciliated cells denuded of cilia in LCC cell layers. Compared with LCC, AIC cell layers (n = 20) achieved a significantly higher peak equivalent short-circuit current (74.1 ± 6.5 vs. 51.6 ± 3.4 µA/cm2), a higher potential difference (70.9 ± 2.8 vs. 60.5 ± 3.0 mV), and a lower peak transepithelial electrical resistance (1.1 ± 0.03 vs. 1.5 ± 0.02 kohms,cm2). About 70% of the short-circuit current in AIC was amiloride-sensitive whereas <10% was furosemide-sensitive, similar to that found in native tissue. The corresponding values in LCC were 50% and 46%. The permeability of both AIC and LCC to lipophilic solutes (dexamethasone and propranolol) was similar, whereas the permeability of hydrophilic solutes (mannitol, sucrose, and albuterol) in AIC was only half that in LCC. Conclusions. Given the striking similarity in morphological and functional characteristics of the AIC to those in the in vivo situation, the AIC is favored as an in vitro model for future drug transport studies.

Inhibition of arachidonic acid release by nordihydroguaiaretic acid and its antioxidant action in rat alveolar macrophages and chinese hamster lung fibroblasts

The ability of nordihydroguaiaretic acid (NDGA) to inhibit arachidonic acid (AA) release from rat alveolar macrophages treated with t-butyl hydroperoxide (tBOOH) or from Chinese hamster lung fibroblasts (V79 cells) treated with linoleic acid hydroperoxide (LOOH) was examined. Treatment of alveolar macrophages with 100 microM tBOOH significantly increased arachidonic acid release and its conversion to metabolites. Pretreatment of macrophages with NDGA (greater than or equal to 2.5 microM) inhibited the release of AA and its subsequent metabolism following addition of tBOOH. Treatment of V79 cells with 1 microM LOOH stimulated the release of AA. Pretreatment with either 1 or 10 microM NDGA prior to the addition of LOOH inhibited the release of AA. A23187 (2 microM)-stimulated release of AA from V79 cells was less sensitive to NDGA inhibition. Pretreatment with 10 microM NDGA, but not with 1 microM NDGA, inhibited A23187-stimulated release of AA. PLA2-dependent hydrolysis of micelle preparations of disaturated phosphatidylcholine was not inhibited by NDGA. Previous studies have suggested that the addition of peroxides alters cells by inducing lipid peroxidation so that the action of phospholipases upon their membranes is enhanced. The results suggest that NDGA, a lipid-soluble antioxidant which traps free radicals, indirectly blocked the action of phospholipases upon cell membranes by inhibiting lipid peroxidation.

Detecting and identifying volatile aldehydes as dinitrophenylhydrazones using gas chromatography mass spectrometry

The detection of aldehydes has become an important measure of lipid oxidation in biological milieu. Aldehyde 2,4-dinitrophenylhydrazones are easily prepared and readily purified by HPLC and/or TLC and have proven useful for the detection of aldehydes. The lower limit of detection for dinitrophenylhydrazones was significantly reduced by using gas chromatography-mass spectrometric (GC-MS) techniques. Individual dinitrophenylhydrazones were readily separated by GC and detected by both positive and negative ion MS. The two major ions in negative ion spectra were the 182 m/z fragment ion and the molecular ion. Positive ion spectra showed strong ions corresponding to the protonated molecular ion and a protonated iminium ion. The greatest sensitivity was obtained with negative ion detection (10 pg per injection). However, more structural information was obtained from analysis of the positive ion spectra. Dinitrophenylhydrazones of hydroxyaldehydes, like 4-hydroxynonenal, were analyzed after converting the dinitrophenylhydrazones into trimethylsiloxylethers. GC-MS with negative ion detection was used to identify and quantitate the release of 4-hydroxynonenal by alveolar macrophages exposed to nitrogen dioxide.

[7] Measurement of γ-glutamyl transpeptidase and γ-glutamylcysteine synthetase activities in cells

Publisher Summary This chapter focuses on the measurement of γ-glutamyl transpeptidase and γ-glutamylcysteine synthetase activities in cells. There has been an increasing interest in glutathione (GSH) as it is the major nonprotein thiol in cells and is a preferred, if not specific, thiol substrate for several enzymes in xenobiotic metabolism and antioxidant defense. Under such stress conditions, cells must maintain glutathione and may even increase glutathione content above the steady state for maximum protection. The intraorgan transport and de novo synthesis of glutathione are two of the possible mechanisms for maintaining or increasing glutathione. The specificity of γGT toward γ-glutamyl compounds is quite broad and has allowed the development of several different assays. The most common assay uses L -γ-glutamyl-p-nitroanilide with the product detected by absorbance spectrophotometry. Several studies have shown that γGT-mediated utilization of extracellular glutathione contributes to protection against oxidative injury. The importance of γGCS in maintaining glutathione in the face of the challenge of even endogenous generation of H 2 O 2 has also been demonstrated.

Release of aldehydes from rat alveolar macrophages exposed in vitro to low concentrations of nitrogen dioxide

This study demonstrated that aldehydes are released into the extracellular medium when alveolar macrophages (AM) are exposed to nitrogen dioxide (NO2) at concentrations that impair cell function but do not cause cell death. Butanal, glycolaldehyde, 4-hydroxynonenal, pentanal, pentenal, and hexanal were found. Dinitrophenylhydrazine (DNP) derivitization, thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry were used to identify the products. Some of the aldehydes have potential toxicity and may be responsible, in part, for altered AM function observed following NO2 exposure.

Depression of stimulated arachidonate metabolism and superoxide production in rat alveolar macrophages following in vivo exposure to 0.5 PPM NO2

Alveolar macrophages (AM) have been found to suffer significant functional deficits in response to nitrogen dioxide (NO2) exposure. The present investigation examined changes in the activation of AM arachidonate metabolism and superoxide production in response to an environmentally relevant level of NO2. Rats were exposed to 0.5 ppm NO2 for periods of 0.5-10 d and AM were obtained by bronchoalveolar lavage (BAL). NO2 exposure produced complex effects upon both unstimulated and stimulated AM arachidonate metabolism. Unstimulated AM synthesis of leukotriene B4 (LTB4) was depressed rapidly within 1 d of exposure, and depressed again at 5 d. Alveolar macrophage production of thromboxane B2 (TxB2), LTB4, and 5-hydroxyeicosatetraenoate (5-HETE) in response to stimulation with the calcium ionophore, A23187, were acutely depressed within 1 d of exposure; however, generation of these compounds recovered to air-control levels with longer exposure, while 5-HETE was increased at 10 d. In contrast, AM production of LTB4 in response to another stimulus, zymosan-activated rat serum (ZAS), was not depressed until following 5 d of exposure and remained slightly lower than air-control levels at 10 d. Levels of TxB2, LTB4, prostaglandin E2 (PGE2), and prostaglandin F2 alpha (PGF2 alpha) measured in BAL fluid (BALF) were found to be depressed within 4 h of exposure, suggesting an acute decrease in the in vivo pulmonary arachidonate metabolism; however, production of these compounds generally recovered to air-control levels with longer exposure. The AM superoxide production stimulated by phorbol myristate acetate (PMA) was decreased rapidly and continuously throughout the study. Thus, exposure to a low concentration of NO2 acutely depresses activation of AM arachidonate metabolism and superoxide production in response to external stimuli, and may impede defense against pulmonary infection.

Dual Effect of Nitrogen Dioxide on Rat Alveolar Macrophage Arachidonate Metabolism

Significant deficits in alveolar macrophage (AM) function have been associated with acute exposure to nitrogen dioxide (NO2). The present investigation examined changes in enzymatic production of arachidonate metabolites from rat AM exposed to NO2. While in vitro exposure of AM to NO2 concentrations between 0.1 and 5 ppm alone had small effects on basal synthesis of cyclooxygenase or lipoxygenase products, exposure to either 1 ppm (2 or 4 h) or 5 ppm (1 h) markedly enhanced the response of AM to stimulation by the calcium ionophore, A23187. This pre-exposure led to significant increases in cyclooxygenase products (thromboxane B2 (thromboxane), the stable metabolite of thromboxane A2, and 12-hydroxyheptadecatrienoic acid (12-HHT)) and lipoxygenase products (leukotriene B (LTB4) and monohydroxyeicosatetraenoate isomers) in response to A23187. In contrast, a 1-h exposure to 20 ppm NO2 alone significantly increased AM synthesis of thromboxane and 12-HHT, but suppressed the effect of subsequently added A23187. Increased synthesis of cyclooxygenase products with 20 ppm NO2 alone were blocked with the phospholipase inhibitor mepacrine and the cyclooxygenase inhibitor indomethacin. The lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) significantly reduced release of arachidonate; however, levels of thromboxane and 12-HHT were significantly increased. The results suggest a dual effect of NO2 on AM arachidonate metabolism in which low concentrations of NO2 had small effects on basal metabolism but markedly amplified the response to stimuli, while a high concentration of NO2 did the reverse. Such a complex dose-response effect may have significant impact in explaining the pathologic effects of NO2.

Air-interface cultures of guinea pig airway epithelial cells: effects of active sodium and chloride transport inhibitors on bioelectric properties

To systematically study airway epithelial barrier properties and active ion transport processes during challenge to environmental toxicants, in vitro models that closely resemble airway epithelium in vivo are required. Guinea pig tracheobronchial epithelial (GPTE) cells cultured in an air interface form a tight, confluent monolayer, which may be a more suitable model for the studies of the airway epithelial barrier in vivo. In the present study, bioelectric properties of such GPTE cell monolayers were characterized with the use of pharmacological agents. Treatment of the basolateral side with ouabain completely abolished the short-circuit current (SCC), while apical addition had little effect. Apical addition of amiloride abolished 75% of the SCC, while basal addition had little effect. Treatment of the basolateral side with furosemide or bumetanide reduced the SCC by 20%, while apical addition had no effect. Apical or basolateral terbutaline increased SCC threefold with identical time courses; results with furosemide suggest a preferential stimulation of Cl- secretion. In contrast to GPTE monolayers, canine tracheal epithelial monolayers, under short-circuit conditions, primarily secrete Cl-, possibly due to apparent species differences. The predominance of Na+ reabsorption suggests that GPTE monolayers cultured in an air interface may share similar transport properties to human airway epithelium.

Dual effect of nitrogen dioxide on barrier properties of guinea pig tracheobronchial epithelial monolayers cultured in an air interface.

Nitrogen dioxide (NO2) is an oxidant gas that may injure the airway epithelial lining, leading to decrements in barrier and active ion transport properties. The present studies examined alterations of bioelectric properties and solute flux by guinea pig tracheobronchial epithelial (GPTE) monolayers exposed in vitro to NO2. Confluent GPTE monolayers were exposed to NO2 levels between 0.5 and 5 ppm, while controls were exposed to air. Following exposure, monolayers were mounted in Ussing chambers for measurement of transepithelial resistance (Rte) and short-circuit current (SCC). A 1-h exposure to 1 ppm NO2 significantly increased SCC to 131.3 +/- 8.7% of air controls, while Rte with a value of 109.3 +/- 13.8% was unchanged. In contrast, a 1-h exposure to 2 or 5 ppm NO2 significantly decreased Rte to 39.0 +/- 1.6 or 35.5 +/- 7.3% of air controls, respectively, while SCC values of 140.3 +/- 10.4 or 153.3 +/- 8.6%, respectively, were also significantly elevated. A 1-h exposure to 2 or 5 ppm NO2 significantly increased sucrose permeability across GPTE monolayers to 446.8 +/- 117 or 313.3 +/- 39.5% of air controls, respectively, while glycerol permeability was unchanged. In contrast, a 1-h exposure to 1 ppm NO2 produced no alterations of sucrose or glycerol flux. The SCC of control GPTE monolayers (1-h air exposure) consisted of 50% bumetanide-sensitive and 40% amiloride-sensitive current; exposure for 1 h to 2 ppm NO2 led to no changes in the corresponding SCC components. Active ion transport (i.e., SCC) across the airway epithelium was significantly increased after exposure to NO2 levels < or = 1 ppm with no change of paracellular pathways for diffusion, suggesting that this reactive gas alters cell membrane function. The increased SCC may lead to impairment of fluid balance and mucociliary clearance. NO2-mediated tissue injury with levels > or = 2 ppm primarily affects passive airway epithelial barrier functions, probably by altering tight junctions, which could result in increased transepithelial solute and fluid leakage in vivo.