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K C Das

Publications and source records attributed to K C Das.

At least 19 recordsLinked to original sources
PubMed2002

Wet scrubber analysis of volatile organic compound removal in the rendering industry.

The promulgation of odor control rules, increasing public concerns, and U.S. Environmental Protection Agency (EPA) air regulations in nonattainment zones necessitates the remediation of a wide range of volatile organic compounds (VOCs) generated by the rendering industry. Currently, wet scrubbers with oxidizing chemicals are used to treat VOCs; however, little information is available on scrubber efficiency for many of the VOCs generated within the rendering process. Portable gas chromatography/mass spectrometry (GC/MS) units were used to rapidly identify key VOCs on-site in process streams at two poultry byproduct rendering plants. On-site analysis was found to be important, given the significant reduction in peak areas if samples were held for 24 hr before analysis. Major compounds consistently identified in the emissions from the plant included dimethyl disulfide, methanethiol, octane, hexanal, 2-methylbutanal, and 3-methylbutanal. The two branched aldehydes, 2-methylbutanal and 3-methylbutanal, were by far the most consistent, appearing in every sample and typically the largest fraction of the VOC mixture. A chlorinated hydrocarbon, methanesulfonyl chloride, was identified in the outlet of a high-intensity wet scrubber, and several VOCs and chlorinated compounds were identified in the scrubbing solution, but not on a consistent basis. Total VOC concentrations in noncondensable gas streams ranged from 4 to 91 ppmv. At the two plants, the odor-causing compound methanethiol ranged from 25 to 33% and 9.6% of the total VOCs (v/v). In one plant, wet scrubber analysis using chlorine dioxide (ClO2) as the oxidizing agent indicated that close to 100% of the methanethiol was removed from the gas phase, but removal efficiencies ranged from 20 to 80% for the aldehydes and hydrocarbons and from 23 to 64% for total VOCs. In the second plant, conversion efficiencies were much lower in a packed-bed wet scrubber, with a measurable removal of only dimethyl sulfide (20-100%).

Air Pollutants↗
PubMed2001

Induction of peroxiredoxin gene expression by oxygen in lungs of newborn primates.

Peroxiredoxin (Prx) is an important antioxidant defense enzyme that reduces hydrogen peroxide to molecular oxygen by using reducing equivalents from thioredoxin. We report that lung Prx I messenger RNA (mRNA) is specifically upregulated by oxygen. Throughout the third trimester, mRNA for Prx I was expressed constitutively at low levels in fetal baboon lung. However, after premature birth (125 or 140 d gestation), lung Prx I mRNA increased rapidly with the onset of oxygen exposure. Premature animals (140 d) breathing 100% O(2) developed chronic lung disease within 7 to 14 d. These animals had greater lung Prx I mRNA after 1, 6, or 10 d of life than did fetal controls. In 140-d animals given lesser O(2) concentrations (as needed) that did not develop chronic lung disease, lung Prx I mRNA also was increased on Days 1 and 6, but not Day 10. In fetal distal lung explant culture, Prx I mRNA was elevated in 95% O(2), relative to 1% oxygen, and remained elevated at 24 h. Prx protein activity increased in 140-d premature baboons exposed to as-needed oxygen. By contrast, there was a decrease in Prx activity in 140-d premature baboons exposed to 100% oxygen. In the lung explants from prematures (140 d), there was no significant increase in Prx activity in response to 24 h exposure to hyperoxia, whereas exposure of explants to 48 h hyperoxia caused a nonsignificant decrease in Prx activity. Treatment of lung explants with actinomycin D inhibited Prx mRNA increases in 95% oxygen, indicating transcriptional regulation. In cellular signaling studies we demonstrated that protein kinase (PK) C activity increased when A549 cells were exposed to 95% oxygen, compared with 21% oxygen exposure. In lung explant cultures, specific PKC inhibitors calphostin C or GF109203X inhibited the increase in Prx I mRNA with 95% oxygen exposure, indicating PKC-mediated signaling. The acute increase in gene expression of Prx I in response to oxygen suggests an important role for this protein during the transition from relatively anaerobic fetal life to oxygen-breathing at birth.

Animals↗
PubMed2000

c-Jun NH2-terminal kinase-mediated redox-dependent degradation of IkappaB: role of thioredoxin in NF-kappaB activation.

NF-kappaB is a redox-sensitive transcription factor known to be activated by oxidative stress as well as chemical and biological reductants. Its DNA binding activity requires reduced cysteines present in the p65 subunit of the dimer. Thioredoxin (Trx) is an endogenous disulfide oxidoreductase known to modulate several redox-dependent functions in the cell. NF-kappaB was activated by addition of Escherichia coli thioredoxin in a redox-dependent manner in A549 cells. Such activation was accompanied by degradation of IkappaB in the cytosol. In addition, only the reduced form of thioredoxin activated NF-kappaB, whereas the oxidized form was without any effect. Overexpression of human thioredoxin also caused activation of NF-kappaB and degradation of IkappaB. On the contrary, dominant-negative redox-inactive mutant thioredoxin expression did not activate NF-kappaB, further confirming the redox-dependent activation of NF-kappaB. We also investigated the mechanism of activation of NF-kappaB by thioredoxin. We demonstrate that thioredoxin activates c-Jun NH(2)-terminal kinase (JNK)-signaling cascade, and dominant-negative expression of mitogen-activated protein kinase kinase kinase 1 (MEKK1), JNK kinase, or JNK inhibits NF-kappaB activation by thioredoxin. In contrast, wild-type MEKK1 or JNK kinase induced NF-kappaB activation alone or in combination with thioredoxin expression plasmid. These findings were also confirmed by NF-kappaB-dependent luciferase reporter gene transcription.

Acetylcysteine↗
PubMed2000

Thioredoxin, a singlet oxygen quencher and hydroxyl radical scavenger: redox independent functions.

Thioredoxin is a ubiquitous small protein known to protect cells and tissues against oxidative stress. However, its exact antioxidant nature has not been elucidated. In this report, we present evidence that human thioredoxin is a powerful singlet oxygen quencher and hydroxyl radical scavenger. Human thioredoxin at 3 microM caused 50% inhibition of TEMP-(1)O(2) (TEMPO) adduct formation in a photolysis EPR study. In contrast, Escherichia coli thioredoxin caused 50% inhibition of TEMPO formation at 80 microM. Both E. coli thioredoxin and human thioredoxin inhibited (*)OH dependent DMPO-OH formation as demonstrated by EPR spectrometry. The quenching of (1)O(2) or scavenging of (*)OH was not dependent upon the redox state of thioredoxin. Using a human thioredoxin in which the structural cysteines were mutated to alanine, Trx-C3A, we show that structural cysteines that do not take part in the catalytic functions of the protein are also important for its reactive oxygen scavenging properties. In addition, using a quadruple mutant Trx-C4A, where one of the catalytic cysteines, C35 was mutated to alanine in addition to the mutated structural cysteines, we demonstrated that catalytic cysteines are also required for the scavenging action of thioredoxin. Identification of thioredoxin as a (1)O(2) quencher and (*)OH scavenger may be of significant importance in explaining various redox-related antioxidant functions of thioredoxin.

Alanine↗
PubMed1999

Effect of oxygen on lung superoxide dismutase activities in premature baboons with bronchopulmonary dysplasia.

We investigated the effects of gestational age and oxygen exposure on superoxide dismutase (SOD) activities in distal fetal lung tissue in primate models of bronchopulmonary dysplasia. During the final third of fetal life, lung coppper-zinc SOD (Cu,ZnSOD) specific activity decreased, whereas lung manganese SOD (MnSOD) specific activity tended to increase. In the premature newborn (140 days, 78% of term gestation), lung total SOD and Cu,ZnSOD specific activities decreased after 6-10 days of ventilation with as needed [pro re nada (PRN)] or 100% oxygen compared with fetal control animals. Neither Cu,ZnSOD mRNA nor protein expression changed after either oxygen exposure at this gestation (140 days) relative to fetal control animals. At this age (6-10 days), lung MnSOD specific activity did not change in oxygen-exposed relative to fetal control animals, even though lung expression of MnSOD mRNA and protein increased after PRN or 100% oxygen exposure. In the very premature 125-day newborn (69% of term), lung Cu,ZnSOD specific activity and protein decreased, whereas Cu,ZnSOD mRNA increased, after 6-10 days of ventilation with PRN oxygen compared with fetal control animals. In fetal lung explants, hyperoxia also decreased expression of SOD activity acutely (16-h exposure, 125 and 140 days gestation). To conclude, expression of SOD activity in the premature primate lung did not increase in response to elevated oxygen tension, apparently due to effects occurring subsequent to the expression of these mRNAs.

Animals↗
PubMed1999

Induction of thioredoxin and thioredoxin reductase gene expression in lungs of newborn primates by oxygen.

Thioredoxin (TRX) is a potent protein disulfide oxidoreductase important in antioxidant defense and regulation of cell growth and signal transduction processes, among them the production of nitric oxide. We report that lung TRX and its reductase, TR, are specifically upregulated at birth by O2. Throughout the third trimester, mRNAs for TRX and TR were expressed constitutively at low levels in fetal baboon lungs. However, after premature birth (125 or 140 of 185 days gestation), lung TRX and TR mRNAs increased rapidly with the onset of O2 or air breathing. Lung TRX mRNA also increased in lungs of term newborns with air breathing. Premature animals (140 days) breathing 100% O2 develop chronic lung disease within 7-14 days. These animals had greater TRX and TR mRNAs after 1, 6, or 10 days of life than fetal control animals. In 140-day animals given lesser O2 concentrations (as needed) who do not develop chronic lung disease, lung TRX and TR mRNAs were also increased on days 1 and 6 but not significantly on day 10. In fetal distal lung explant culture, mRNAs for TRX and TR were elevated within 4 h in 95% O2 relative to 1% O2, and the response was similar at various gestations. In contrast, TRX protein did not increase in lung explants from premature animals (125 or 140 days) but did in those from near-term (175-day) fetal baboons after exposure to hyperoxia. However, lung TRX protein and activity, as well as TR activity, eventually did increase in vivo in response to hyperoxia (6 days). Increases in TRX and TR mRNAs in response to 95% O2 also were observed in adult baboon lung explants. When TRX redox status was determined, increased O2 tension shifted TRX to its oxidized form. Treatment of lung explants with actinomycin D inhibited TRX and TR mRNA increases in 95% O2, indicating transcriptional regulation by O2. The acute increase in gene expression for both TRX and TR in response to O2 suggests an important role for these proteins during the transition from relatively anaerobic fetal life to O2 breathing at birth.

Air↗
PubMed1998

Protein kinase Cdelta-dependent induction of manganese superoxide dismutase gene expression by microtubule-active anticancer drugs.

Bacterial lipopolysaccharide can induce manganese superoxide dismutase (MnSOD) gene expression in a variety of cells. Paclitaxel (taxol) shares many properties of lipopolysaccharide. Here we report that paclitaxel can induce MnSOD gene expression in human lung adenocarcinoma cell line A549 in a time- and dose-dependent manner. Additional anticancer drugs, vinblastine and vincristine, also induced MnSOD gene expression. We have shown previously (Das, K. C., and White, C. W. (1997) J. Biol. Chem. 272, 14914-14920) that these drugs can activate protein kinase C (PKC). The PKC agonists thymeleatoxin (0.5 microM) and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA; 10 nM) potently induced MnSOD gene expression. Calphostin C and GF109203X, both specific inhibitors of PKC, each inhibited MnSOD gene expression by anticancer agents. Down-regulation of PKC by prolonged treatment with phorbol 12-myristate 13-acetate (PMA) also inhibited induction of MnSOD by anticancer drugs, indicating an important role of PKC in MnSOD signaling by these agents. Of 11 PKC isoenzymes, only PKCdelta translocated to the cell membrane after stimulation with anticancer drugs. By contrast, dPPA, PMA, and thymeleatoxin caused translocation of PKCalpha, betaI, delta, and mu isotypes. Anticancer drug-stimulated cells also had increased total PKC activity in membrane and cytosolic fractions. Thus, paclitaxel, vinblastine, and vincristine each specifically activate PKCdelta, whereas PMA, thymeleatoxin, and dPPA activate multiple isoenzymes. PKCdelta was the only isoform activated by each agent in both groups of compounds effective in MnSOD induction.

Antineoplastic Agents, Phytogenic↗
PubMed1998

Detection of thioredoxin in human serum and biological samples using a sensitive sandwich ELISA with digoxigenin-labeled antibody.

Thioredoxin is a low molecular weight, redox active protein important in cellular proliferation, signal transduction and antioxidant function. Thioredoxin is secreted by normal as well as neoplastic cells and is potentially involved in paracrine cell communication as suggested by its co-cytokine activity. Thus, the thioredoxin level in biological fluids, cells and tissue homogenates could be an important indicator of physiological or pathophysiological conditions. Hence, an accurate and sensitive measurement is of paramount importance in studies involving thioredoxin. We present here an ultrasensitive enzyme linked immuno-absorbent assay (ELISA) for human thioredoxin using digoxigenin-labelled goat polyclonal anti-human thioredoxin. The assay could detect a minimum level of 15 pg/ml thioredoxin in human serum, cell culture media, and in cell and tissue samples. The assay was optimized for concentration of both antibodies, blocking agent, plates, incubation time and reaction volumes. Excellent linearity and reproducibility were obtained. The assay was applied to different baboon tissues and human serum samples. The intrassay coefficient of variation (CV) was between 6.0 to 14 and the interassay CV was from 1.6 to 11.1. Excellent parallelism of standards with serum samples, tissue homogenates or cell lysates was obtained. More than 90% recovery of human thioredoxin was observed in 10% human serum. The assay is easy to use, rapid, reproducible, but above all it is a quantitative, specific and sensitive way to measure thioredoxin in a variety of biological specimens.

Animals↗
PubMed1998

Cytokine-induced nitric oxide formation in normal but not in neoplastic murine lung epithelial cell lines.

Cytomix, a mixture of interferon-gamma, tumor necrosis factor-alpha, and interleukin-1beta, induces nitric oxide (NO) production in lung epithelial cell lines. It is not known whether neoplastic transformation alters a cell's ability to form NO in response to cytokines. The present study investigated NO formation in two murine lines of immortalized "normal" (nontumorigenic) lung epithelial cells of alveolar type II origin, E10 and C10, and their sibling spontaneous transformants, E9 and A5. Nontumorigenic cells elaborated much more NO after cytomix exposure than did their tumorigenic counterparts. NO production was prevented by inhibiting protein synthesis and NO synthase and attenuated by dexamethasone. Northern and Western blot analyses of inducible NO synthase (iNOS) demonstrated cytomix-induced induction of iNOS only in nontumorigenic cells. The deficiency in NO production in tumorigenic cells was not associated with reduced iNOS mRNA stability or with differences in cytomix-induced nuclear factor-kappaB activation. Although cytomix caused a greater production of NO in E10 cells than in E9 cells, the same treatment induced equivalent proliferation in both cell lines. These results indicate a specific deficiency in cytokine-induced NO synthesis in transformed murine lung epithelial cells relative to their normal progenitor cells and provide a model for investigating iNOS regulation.

Animals↗
PubMed1997

Activation of NF-kappaB by antineoplastic agents. Role of protein kinase C.

Paclitaxel can induce tumor necrosis factor (TNF) and interleukin-1 gene expression, similar to lipopolysaccharides. Since lipopolysaccharide-induced expression of TNF is related to activation of NF-kappaB, we determined whether NF-kappaB could be activated by paclitaxel. In the human lung adenocarcinoma cell line A549, paclitaxel activated NF-kappaB in a dose-dependent manner with maximal activation after 2-4 h. Since paclitaxel could up-regulate TNF and interleukin-1 secretion and subsequent NF-kappaB activation could be caused by these cytokines, the effect of two other groups of anticancer drugs including vinca alkaloids (vinblastine and vincristine) and anthracyclines (daunomycin and doxorubicin), neither of which induce TNF or interleukin-1 gene expression, were examined. Like paclitaxel, vinblastine, vincristine, daunomycin, and doxorubicin each caused activation of NF-kappaB. Therefore, it is unlikely that activation of NF-kappaB caused by these agents or by paclitaxel is mediated via cytokine up-regulation. Furthermore, actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively, did not inhibit paclitaxel-induced NF-kappaB activation. Several other transcription factors such as AP-1, AP-2, CREB, SP-1, or TFIID were not activated by antineoplastic agents demonstrating specificity of NF-kappaB activation. The involvement of both subunits in the NF-kappaB DNA binding complex was demonstrated by its abrogation by anti-p65 and by supershift by anti-p50 antibodies. Since protein phosphorylation is implicated in the activation of NF-kappaB, the effect of anticancer drugs on protein kinase C activity was measured. Vincristine, daunomycin, and paclitaxel significantly increased protein kinase C activity, and vinblastine and doxorubicin caused similar trends. Following treatment with antineoplastics (1-4 h), cytoplasmic IkappaBalpha degradation occurred concomitantly with translocation of p65 to the nucleus. Specific protein kinase C inhibitors (bisindolylmaleimide (GF109203X) and calphostin C) blocked the activation of NF-kappaB by each compound. Hence, protein kinase C activation may contribute to NF-kappaB activation by antineoplastic agents.

Adenocarcinoma↗
PubMed1997

Elevation of manganese superoxide dismutase gene expression by thioredoxin.

Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that dismutates potentially toxic superoxide radical into hydrogen peroxide and dioxygen. This enzyme is critical for protection against cellular injury due to elevated partial pressures of oxygen. Thioredoxin (TRX) is a potent protein disulfide reductase found in most organisms that participates in many thiol-dependent cellular reductive processes and plays an important role in antioxidant defense, signal transduction, and regulation of cell growth and proliferation. Here we describe induction of manganese superoxide dismutase by thioredoxin. MnSOD mRNA and activity were increased dramatically by low concentrations of TRX (28 microM). Elevation of MnSOD mRNA by TRX was inhibited by actinomycin D, but not cycloheximide, occurring both in cell lines and primary human lung microvascular endothelial cells. mRNAs for other antioxidant enzymes including copper-zinc superoxide dismutase and catalase were not elevated, demonstrating specificity of induction of MnSOD by TRX. Thiol oxidation by diamide or alkylation by chlorodinitrobenzene inhibited MnSOD induction, further indicating a requirement for reduced TRX. Because both oxidized and reduced thioredoxin (28 microM) induced MnSOD mRNA, the intracellular redox status of externally added Escherichia coli oxidized TRX was determined. About 45% of internalized E. coli TRX was reduced, with 8% in fully reduced form and about 37% in partially reduced form. However, when TRX reductase and nicotinamide adenine dinucleotide (NADPH) were added to the extracellular medium with TRX, more than 80% of E. coli TRX was found to be in a fully reduced state in human adenocarcinoma (A549) cells. Although lower concentrations of oxidized TRX (7 microM) did not induce MnSOD mRNA, this concentration of TRX, when reduced by NADPH and TRX reductase, increased MnSOD mRNA six-fold. In additional studies, MCF-7 cells stably transfected with the human TRX gene had elevated expression of MnSOD mRNA relative to vector-transfected controls. Thus, both endogenously produced and exogenously added TRX elevate MnSOD gene expression. These findings suggest a novel mechanism involving reduced TRX in regulation of MnSOD.

Blotting, Western↗
PubMed1997

Pathophysiology of bleeding in heat stress: an experimental study in sheep.

Widespread hemorrhagic manifestations commonly occur in patients with severe heat stroke. The pathogenesis of hemostatic disorders in these patients is not fully understood, although it is believed to be multifactorial in origin. The present investigation was designed to study the changes in blood platelets caused by heat stress in an experimental model of five merino sheep. The experiments were performed in two groups of five merino sheep each. In one group the sheep were subjected to a combination of heat (elevated environmental temperature) and exertional stress, and allowed to proceed throughout the experiment until a state of near collapse was reached (Task A). In the other group (Task B) the animals were heated in the same manner as those in Task A and also subjected to exertional heat; however, when the temperature reached 43.6 +/- 0.2 degrees C, the critical core temperature (CCT), they were subjected to evaporative cooling in a climatic chamber. Serial changes in the platelet counts and platelet functions were measured throughout the duration of the experiments. At the core temperature (CT) of 42.1 degrees C and above there was a significant impairment of adhesion of platelets to glass beads. During the early phases of elevation of CT, platelets showed hyperaggregation in the presence of different agonists (such as, collagen, ADP, ristocetin); this was followed by hypoaggregation when the CCT was raised above 43.6 +/- 0.2 degrees C. However, these impairments of platelet functions occurring at elevated CT and CCT were found to reverse to normal within 24 hours after the animals were cooled to 39 degrees C. It was also found that the hyperaggregation of platelets to different agonists induced by raised CT could be partially prevented by prior in vitro treatment of platelets with apyrase, a known enzyme destroying of ADP. The results of these experiments indicate that heat stress induced by exposing merino sheep to elevated controlled temperature directly activates the platelets. This may be an important contributing factor in causing altered hemostasis in heat stroke activated directly by heat. This mechanism may be operating in altered hemostasis in heat stroke.

Animals↗
PubMed1996

Thrombophilia in ethnic Arabs in Kuwait.

One hundred and thirty unrelated patients with recurrent deep venous thrombosis were studied over a period of 4 years (1986-1990) in order to determine the possible etiology. Protein C levels were estimated in plasma both by chromogenic substrate assay and by immunoassay. Protein S levels in plasma was determined by immunoassay using antisera to human protein S. Antithrombin III (AT-III) was assayed using monospecific rabbit antiserum to human AT-III. Fifteen patients were found to have hereditary protein C deficiency (11.52%). Family studies revealed autosomal recessive inheritance in one patient and a dominant pattern in the remaining 14 patients. Protein S deficiency was found in eight cases (6.1%), AT-III deficiency was established in five cases (3.8%) and a fibrinolytic defect in 33 cases (25.4%). Thrombosis of visceral and cerebral vessels and a positive family history were more frequently found among patients who had hereditary deficiency of one or the other antithrombotic factor. Thrombophlebitis of superficial veins was found to be very common in patients with protein C and protein S deficiency and virtually absent in AT-III deficiency. The high frequency of protein C and protein S deficiency in this ethnic group is attributed to the high frequency of consanguinity.

ABO Blood-Group System↗
PubMed1995

Thiol modulation of TNF alpha and IL-1 induced MnSOD gene expression and activation of NF-kappa B.

TNF alpha and IL-1 each can activate NF-kappa B and induce gene expression of manganese superoxide dismutase (MnSOD), a mitochondrial matrix enzyme which can provide critical protection against hyperoxic lung injury. The regulation of MnSOD gene expression is not well understood. Since redox status can modulate NF-kappa B and potential kappa B site(s) exist in the MnSOD promoter, the effect of thiols (including NAC, DTT and 2-ME) on TNF alpha and IL-1 induced activation of NF-kappa B and MnSOD gene expression was investigated. Activation of NF-kB and increased MnSOD expression were potentiated by thiol reducing agents. In contrast, thiol oxidizing or alkylating agents inhibited both NF-kappa B activation and elevated MnSOD expression in response to TNF alpha or IL-1. Since protease inhibitors TPCK and TLCK can inhibit NF-kappa activation, we also investigated the effect of these compounds on MnSOD expression and NF-kappa B activation. TPCK and TLCK each inhibited MnSOD gene expression and NF-kappa B activation. Since the MnSOD promoter also contains an AP-1 binding site, the effect of thiols and thiol modifying agents on AP-1 activation was investigated. Thiols had no consistent effect on AP-1 activation. Likewise, some of the thiol modifying compounds inhibited AP-1 activation by TNF alpha or IL-1, whereas others did not. Since diverse agents had similar effects on activation of NF-kappa B and MnSOD gene expression, we have demonstrated that activation of NF-kappa B and MnSOD gene expression are closely associated and that reduced sulfhydryl groups are required for cytokine mediation of both processes.

Adenocarcinoma↗
PubMed1995

Activation of NF-kappa B and elevation of MnSOD gene expression by thiol reducing agents in lung adenocarcinoma (A549) cells.

The effect of reducing agents, including N-acetylcysteine (NAC), dithiothreitol (DTT), and 2-mercaptoethanol (2-ME) on nuclear transcription factor-kappa B (NF-kappa B) activation and manganese superoxide dismutase (MnSOD) expression was investigated in a pulmonary adenocarcinoma (A549) cell line. NAC, DTT, and 2-ME each activated the transcription factor NF-kappa B and increased steady-state levels of MnSOD mRNA and enzyme activity in these cells. In addition, NAC, DTT, and 2-ME increased chloramphenicol acetyltransferase (CAT) activity in cells transfected with a construct containing the CAT gene under the control of the rat MnSOD promoter. SOD and catalase (500 U/ml) plus ethanol (1 mM) did not inhibit activation of NF-kappa B or elevation of steady-state MnSOD mRNA levels by NAC, DTT, or 2-ME. Controls in which comparable amounts of O2-. to those produced by thiols were generated by hypoxanthine and xanthine oxidase, or in which H2O2 was added directly, had neither activated NF-kappa B nor elevated MnSOD mRNA. This shows that reactive oxygen intermediates, which may be formed during autooxidation, may not contribute to activation of NF-kappa B. Because the MnSOD promoter also contains potential binding sites for other transcription factors, such as promoter-selective transcription factor-1 (SP-1), activator protein-1 (AP-1), AP-2, adenosine 3',5'-cyclic monophosphate-regulator element binding factor (CREB), and transcription factor IID complex (TFIID), the effect of thiols on their activation also were evaluated. In contrast to findings with NF-kappa B, there was only minor activation of AP-1 by thiols, and none of the other transcription factors were activated by thiols. AP-1 activation was inhibited by catalase (500 U/ml) plus SOD plus ethanol (1 mM). Addition of 700 microM H2O2 also activated AP-1, and catalase at 500 U/ml prevented this activation. This indicates that H2O2 produced as a result of autooxidation of thiols can activate AP-1 but not NF-kappa B. Thus a close association between exposure to reducing agents, activation of NF-kappa B, and elevation of MnSOD gene expression is demonstrated.

Acetylcysteine↗
PubMed1994

Impairment of raw 264.7 macrophage function by antiarrhythmic drugs.

The effect of the antiarrhythmic drugs lidocaine, quinidine and procainamide on macrophage function was investigated in RAW 264.7 mouse monocytic macrophage cell. Cells stimulated by either zymosan or phorbol ester were found to generate both superoxide (O2-) and H2O2. The production of O2 was detected as superoxide dismutase inhibitable ferricytochrome c reduction. H2O2 production was monitored in both chemical and flow cytometric fluorescent assays. Although all three drugs inhibited both O2 and H2O2 release in a dose-dependent manner, only quinidine was found to have significant inhibitory effects. The amounts of quinidine required to cause a 50% inhibition in O2 production in zymosan and phorbol ester stimulated cells were found to be 250 microM and 300 microM, respectively and the amounts required to cause one-half optimum levels of H2O2 production in these cells were found to be 50 microM and 100 microM, respectively. The effect of these drugs on O2 producing NADPH oxidase was investigated and only procainamide was found to have a significant effect (p < 0.001) in inhibiting the oxidase activity. Lidocaine and quinidine had no significant effect on the activation of the respiratory burst oxidase. A sensitive and convenient 'differential phagocytosis' assay was devised on the basis of number of particles engulfed by individual phagocytes using flow cytometric techniques. It appears to be remarkably free of interference and was applied to investigate the role of antiarrhythmic drugs on the phagocytosis of fluorescent latex beads. All three antiarrhythmic drugs inhibited phagocytosis of latex beads in a dose dependent manner irrespective of the number of particles phagocitized by the cells. The results of these studies do not conclusively establish a mechanism of action of these drugs on the generation of O2 and H2O2 by stimulated macrophages; nevertheless, it is interesting that all three drugs inhibited the phagocytic activity.

Animals↗