Effects of superoxide dismutase and polyclonal tumor necrosis factor-alpha antibodies on chloroacetate-induced cellular death and superoxide anion production by J774.A1 macrophages

Dichloroacetate (DCA) and trichloroacetate (TCA) are by-products that are formed during the process of water chlorination and have been previously shown to induce superoxide anion (SA) production and cellular death when added to J774.A1 macrophage cultures. In this study, the effects of superoxide dismutase (SOD) and polyclonal tumor necrosis factor-alpha (TNF-alpha) antibodies on DCA- and TCA-induced SA production and cellular death have been tested on the J774.A1 macrophage cultures. TCA and DCA were added to different cultures either alone, each at a concentration of 16 mM, or in combination with SOD (2-12 units/ml), or with TNF-alpha antibodies (10 and 25 units/ml). Cells were incubated for 48 h, after which cellular death/viability, lactate dehydrognase (LDH) leakage by the cells, and SA production by the cells were determined. While TCA and DCA caused significant cellular toxicity, indicated by reduction in cellular viability and increases in LDH leakage and SA production, SOD addition resulted in significant reduction of the effects induced by the compounds. On the other hand, addition of TNF-alpha antibodies to the DCA- and TCA-treated cultures resulted in significant reduction of DCA- but not TCA-induced cellular death and SA production by the cells. Although these results suggest a significant role for SA in DCA- and TCA-induced cellular death, they may also suggest two different mechanisms for the chloroacetate-induced SA production by the cells.     

The induction of tumor necrosis factor‐alpha,superoxide anion,myeloperoxidase, and superoxide dismutase in the peritoneal lavage cells of mice after prolonged exposure to dichloroacetate and trichloroacetate

The induction of phagocytic activation in response to prolonged treatment with different doses of dichloroacetate (DCA) and trichloroacetate (TCA) has been investigated in mice. Groups of B6C3F1 male mice were administered 7.7, 77, 154, and 410 mg of DCA or TCA/kg/day, postorally, for 4‐ and 13‐weeks. Peritoneal lavage cells (PLCs) were isolated and assayed for the different biomarkers of phagocytyic activation, including superoxide anion (SA), tumor necrosis factor‐alpha (TNF‐α), and myeloperoxidase (MPO). In addition, the role of superoxide dismutase (SOD) in the SA production was also assessed. DCA and TCA produced significant and dose‐dependent increases in SA and TNF‐α production and in MPO activity, but the increases in response to the high doses of the compounds (>77 mg/kg/day) in the 13‐week treatment period were less significant than those produced in the 4‐week treatment period. Also, dose‐dependent increases in SOD activity were observed in both periods of treatments. In general, the results demonstrate significant induction of the biomarkers of phagocytic activation by doses of DCA and TCA that were previously shown to be noncarcinogenic, with significantly greater increases observed at the earlier period of exposure, as compared with later period. These findings may argue against the contribution of those mechanisms to the hepatotoxicity/hepatocarcinogenicity of the compounds and suggest them to be early adaptive/ protective mechanisms against their long‐term effects. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:136–144, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20322     

Dichloroacetate and Trichloroacetate Toxicity in AML12 Cells: Role of Oxidative Stress

The toxicity of the drinking water disinfection by products dichloroacetate (DCA) and trichloroacetate (TCA) was studied in the alpha mouse liver (AML12) cells at concentrations ranging between 770 and 4100 ppm and at incubation times ranging from 24 to 72 h. Cellular viability, superoxide anion (SA) and lipid peroxidation (LP) production, as well as superoxide dismutase (SOD) activity were determined. DCA and TCA resulted in time‐ and concentration‐dependent decreases in cellular viability, and also in significant increases in SA and LP production, and in SOD activity at specific concentrations and time points. The effective toxic concentrations of the compounds in these cells were found to be 10‐fold higher than those producing similar effects in the mouse liver. It has been concluded that the AML12 is a good screening system to identify toxic concentrations of the halaocetates present in the drinking water that may need further in vivo testing.     

Comparative toxicity studies on bromochloroacetate,dibromoacetate, and bromodichloroacetate in J774A.1 macrophages: Roles of superoxide anion and protein carbonyl compounds

The brominated and mixed bromo‐chloro‐haloacetates, such as dibromoacetate (DBA), bromochloroacetate (BCA), and bromodichloroacetate (BDCA), are by‐products of water chlorination and are found at lower levels than the fully chlorinated acetates in the drinking water. The toxicities of the compounds were assessed in J774A.1 cells and were found to induce concentration‐dependent increases in cell death and superoxide anion and protein carbonyl compounds production. Compared to the previously tested concentrations of dichoroacetate (DCA) and trichloroacetate (TCA) in the same cell line, the tested haloacetates induced similar effects on cellular viability and superoxide anion production but at DBA and BCA concentrations that were approximately 40–160 times lower than those of DCA and TCA, and at BDCA concentrations that were 4–16 times lower than those of DCA and TCA. Also, production of super oxide anion, protein carbonyl compounds, and induction of phagocytic activation are suggested to play a role in their toxicity.     

Chromium-induced production of reactive oxygen species,DNA single-strand breaks,nitric oxide production,and lactate dehydrogenase leakage in J774A.1 cell cultures

The involvement of oxidative stress in the toxicity of chromium (VI) and chromium (III) has been proposed. We have therefore examined the effects of these cations on the production of superoxide anion, nitric oxide (NO), and DNA single strand breaks (SSB) in J774A.1 macrophage cells in culture as well as the effects on lactate dehydrogenase (LDH) leakage and cell viability. Following a 48 hour incubation, over twofold increases in superoxide anion and NO production were observed at concentrations of approximately 0.30 and 50 μM for Cr (VI) and Cr (III), respectively. The patterns of cell viability and LDH leakage paralleled superoxide anion and NO production for Cr (VI) and Cr (III). A 50% decrease in viability was observed at approximately the concentrations that produced a twofold increase in superoxide and NO production. Concentration-dependent increases in DNA-SSB were observed after incubation with Cr (III) with maximum increases occurring at a concentration of approximately 60 μM. Cr (VI) had no effect on the incidence of DNA-SSB at any of the tested concentrations. The results indicate that Cr (VI) and Cr (III) are toxic to the J774A.1 cell line, and the toxicity may be due at least in part to an oxidative stress induced by the production of reactive oxygen species. © 1996 John Wiley & Sons, Inc.     

Dichloroacetate- and trichloroacetate-induced phagocytic activation and production of oxidative stress in the hepatic tissues of mice after acute exposure

Dichoroacetate (DCA) and trichloroacetate (TCA) are by-products formed during chlorination of the drinking water and were found to be hepatotoxic and hepatocarcinogenic in rodents. In this study, the abilities of the compounds to induce oxidative stress and phagocytic activation have been studied in B6C3F1 mice. Groups of mice were administered 300 mg/kg of either DCA or TCA, p.o, and were sacrificed after 6 or 12 h. Peritoneal lavage cells (PLCs) were isolated and assayed for superoxide anion (SA) production, and hepatic tissues were assayed for the production of SA, lipid peroxidation (LP), and DNA-single strand breaks (SSBs). TCA resulted in significant production of SA in the PLCs, and in the production of SA, LP, and DNA-SSBs in the hepatic tissues, 12 h after dosing, as compared with the control. DCA administration, on the other hand, resulted in significant increases in the productions of LP and DNA-SSBs in the hepatic tissues at both time points, and in SA production in PLCs and hepatic tissues, 6 h after dosing. However, DCA-induced increases in SA production in PLC and hepatic tissues declined at the 12-h time point, reaching control level in the hepatic tissues. These results may implicate the contribution of phagocytic activation to the induction of oxidative stress in the hepatic tissues and also the role of SA production in the induction of LP and/or DNA damage in those tissues, in response to the compounds. The results also suggest studying the involvement of these mechanisms in the long-term hepatotoxicity/hepatocarcinogencity of the compounds.     

Effect of oxygen and endotoxin on lactate dehydrogenase release, 5-hydroxytryptamine uptake, and antioxidant enzyme activities in endothelial cells

We compared the effects of 95% O2 (hyperoxia) alone, endotoxin (20 ng/ml) alone, and 95% O2 plus endotoxin on the release of lactate dehydrogenase (LDH), uptake of 5-hydroxytryptamine (5-HT), and antioxidant enzyme activities in porcine pulmonary arterial and aortic endothelial cells in monolayer culture. Hyperoxia increased LDH release and decreased 5-HT in both endothelial cell types. Hyperoxia also caused a decrease in catalase (CAT) activity and an increase in total superoxide dismutase (SOD) and glutathione reductase (GSH-Red) activities in both cell types. Endotoxin alone had no effect on LDH release, 5-HT uptake, or antioxidant enzyme activities. However, endotoxin prevented the hyperoxic increase in LDH release and the hyperoxic decrease in 5-HT uptake. Endotoxin plus 95% O2 had no consistent effect on the antioxidant enzyme profile in pulmonary artery or aortic endothelial cells. These results indicate that (1) hyperoxia injures both pulmonary artery and aortic endothelial cells in culture and causes changes in the antioxidant enzyme profile that are similar in the two cell types; (2) hyperoxia-induced decreases in CAT activity and increases in SOD activity may be responsible for increased sensitivity of endothelial cells to O2 toxicity; and (3) endotoxin protects against hyperoxic injury to endothelial cells in vitro, but increases in antioxidant enzyme activities are not the mechanism for this protection.     

Ricin-induced toxicity in the macrophage J744A.1 cells: the role of TNF-alpha and the modulation effects of TNF-alpha polyclonal antibody

Ricin is a natural toxin of the castor beans (Ricinus communus). We studied the time- and concentration-dependent effects of ricin on the release of TNF-alpha and lactate dehydrogenase (LDH), as well as the modulation of the ricin-induced effects by TNF-alpha antibody in the J774A.1 cells. When added at concentrations ranging from 0 to 1000 ng/mL, ricin caused concentration-dependent increases in the release of TNF-alpha after incubation for 12 to 24 hours. Concentration-dependent increases in the leakage of LDH were also observed after incubation of the cells with those concentrations of ricin for 24 to 48 hours. Addition of 5 units/mL of rabbit anti-mouse TNF-alpha polyclonal antibody (TNF-alpha antibody) 2 hours prior to the addition of ricin resulted in a decrease in the ricin-induced toxicity, indicated by the release of LDH by the cells. However, when added at concentrations higher than 5 units/mL, the antibody resulted in either no effect or an increase in the ricin-induced LDH leakage. These results suggest that secretion of TNF-alpha by the macrophages in response to ricin plays a significant role in the toxicity of ricin and that TNF-alpha antibody can antagonize the effects of ricin in this cell line when added at relatively low concentrations.     

Fc-receptor-mediated intracellular delivery of Cu/Zn-superoxide dismutase (SOD1) protects against redox-induced apoptosis through a nitric oxide dependent mechanism

BACKGROUND: Using specific antibodies against bovine Cu/Zn-superoxide dismutase (EC 1.15.1.1, SOD1) we demonstrated that anti-SOD antibodies (IgG1) are able to promote the intracellular translocation of the antioxidant enzyme. The transduction signalling mediated by IgG1 immune complexes are known to promote a concomitant production of superoxide and nitric oxide leading to the production of peroxynitrites and cell death by apoptosis. The Fc-mediated intracellular delivery of SOD1 thus limited the endogenous production of superoxide. It was thus of interest to confirm that in the absence of superoxide anion, the production of nitric oxide protected cells against apoptosis. Study in greater detail clearly stated that under superoxide anion-free conditions, nitric oxide promoted the cell antioxidant armature and thus protected cells against redox-induced apoptosis. MATERIALS AND METHODS: The murine macrophage cell-lines J774 A1 were preactivated or not with interferon-gamma and were then stimulated by IgG1 immune complexes (IC), free SOD1 or SOD1 IC and superoxide anion, nitric oxide, peroxynitrite, and tumor necrosis factor-alpha (TNF-alpha) production was evaluated. The redox consequences of these activation processes were also evaluated on mitochondrial respiration and apoptosis as well as on the controlled expression of the cellular antioxidant armature. RESULTS: We demonstrated that SOD1 IC induced a Fcgamma receptor (FcgammaR)-dependent intracellular delivery of the antioxidant enzyme in IFN-gamma activated murine macrophages (the J774 AI cell line). The concomitant stimulation of the FcyR and the translocation of the SOD1 in the cytoplasm of IFN-gamma-activated macrophages not only reduced the production of superoxide anion but also induced the expression of the inducible form of nitric oxide synthase (iNOS) and the related NO production. This inducing effect in the absence of superoxide anion production reduced mitochondrial damages and cell death by apoptosis and promoted the intracellular antioxidant armature. CONCLUSIONS: To define the pharmacologic mechanism of action of bovine SOD1, we attempted to identify the second messengers that are induced by SOD1 IC. In this work, we propose that Fc-mediated intracellular delivery of the SOD1 that reduced the production of superoxide anion and of peroxynitrite, promoted a NO-induced protective effect in inducing the antioxidant armature of the cells. Taken together, these data suggested that specific immune responses against antigenic SOD1 could promote the pharmacological properties of the antioxidant enzyme likely via a NO-dependent mechanism.     

Toxicological effects of iron on intestinal cells

The aim of the present study was to investigate whether iron, which is involved in the formation of free radicals in vitro, can initiate cellular injury in human intestinal cells. The effects of various concentrations of iron were studied in preconfluent, colonic-cancerogenous cells, and also in postconfluent, differentiating cells. Cellular damage was assessed using cell proliferation (serial cell counting), tetrazolium dye (MTT) uptake, lactate dehydrogenase (LDH) release and apoptosis studies based on caspase-3 activities. Also the activities of the major antioxidative enzymes, superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were measured after the cells had been exposed to iron. Our results indicated that preconfluent cells were more susceptible to iron toxicity, as assessed by a significant reduction in cell proliferation and MTT uptake in a concentration-dependent manner compared to the control. However, no evidence for MTT uptake was observed in postconfluent cells. Caspase-3 activity, an indicator of cell apoptosis, considerably increased in preconfluent cells at high iron levels compared to the control (p < 0.05), whereas postconfluent cells were not significantly affected. LDH release was similar for both groups and was significantly higher than the control at 900 microM iron and above. SOD activities were not affected by iron in either group, whereas GPx was considerably higher in iron-treated cells in both groups compared with the control (because of relatively high standard deviations this effect was not significant). In conclusion we suggest that iron exerts its toxic effects intracellularly especially in preconfluent Caco-2 cells, whereas only high iron doses were able to alter the viability of differentiating, enterocyte-like cells.     

Time- and concentration-dependent production of superoxide anion, nitric oxide, DNA damage and cellular death by ricin in the J774A.1 macrophage cells

The time- and concentration-dependent effects of ricin on some biomarkers of cellular toxicity, including production of superoxide anion (O2-), nitric oxide (NO), and DNA single strand breaks (SSB), as well as cellular death, have been examined in the J774A.1 macrophage cell cultures. Various concentrations of ricin have been added to various cell cultures, and the cells were incubated for 12, 24, 36, and 48 hours. Following 12 hour incubation, ricin did not cause significant increases in any of those biomarkers. However, time- and concentration-dependent increases were observed in the induction of all the biomarkers after incubation for 24-48 hours. Approximately twofold increases in the production of O2- were observed after incubation with 1 and 10 ng/mL of ricin for 24 and 36-48 hours, respectively. The concentrations of ricin that caused approximately twofold increases in the rate of DNA-SSB are 10 and 1-10 ng/mL after 24 and 36-48 hours incubation, respectively. Approximately twofold increases in NO production were only observed after incubation of the cultures with 1-10 ng/mL of ricin for 36-48 hours. Fifty percent reductions in cellular viability were also observed with ricin concentrations of 10-100, 10, and 1-10 ng/mL, after incubation for 24, 36, and 48 hours, respectively.     

Superoxide Triggers an Acid Burst in Saccharomyces cerevisiae to Condition the Environment of Glucose-starved Cells

Although yeast cells grown in abundant glucose tend to acidify their extracellular environment, they raise the pH of the environment when starved for glucose or when grown strictly with non-fermentable carbon sources. Following prolonged periods in this alkaline phase, Saccharomyces cerevisiae cells will switch to producing acid. The mechanisms and rationale for this “acid burst” were unknown. Herein we provide strong evidence for the role of mitochondrial superoxide in initiating the acid burst. Yeast mutants lacking the mitochondrial matrix superoxide dismutase (SOD2) enzyme, but not the cytosolic Cu,Zn-SOD1 enzyme, exhibited marked acceleration in production of acid on non-fermentable carbon sources. Acid production is also dramatically enhanced by the superoxide-producing agent, paraquat. Conversely, the acid burst is eliminated by boosting cellular levels of Mn-antioxidant mimics of SOD. We demonstrate that the acid burst is dependent on the mitochondrial aldehyde dehydrogenase Ald4p. Our data are consistent with a model in which mitochondrial superoxide damage to Fe-S enzymes in the tricarboxylic acid (TCA) cycle leads to acetate buildup by Ald4p. The resultant expulsion of acetate into the extracellular environment can provide a new carbon source to glucose-starved cells and enhance growth of yeast. By triggering production of organic acids, mitochondrial superoxide has the potential to promote cell population growth under nutrient depravation stress.     

Salicylic acid induces apoptosis in colon carcinoma cells grown in-vitro: influence of oxygen and salicylic acid concentration

In solid tumors the hypoxic environment can promote tumor progression and resistance to therapy. Recently, acetylsalicylic acid a major component of analgesic drugs and its metabolite salicylic acid (SA) have been shown to reduce the risk of colon cancer, but the mechanisms of action remain still unclear. Here we elucidate the effects of physiologically relevant concentrations of SA on colon carcinoma cells (CaCo-2) grown under normoxic and hypoxic conditions. Western blotting, caspase-3/7 apoptosis assays, MTS cell-proliferation assays, LDH cytotoxicity assays and hydrogen peroxide measurements were performed to investigate the effects of 1 and 10μM SA on CaCo-2 cells grown under normoxic conditions and cells exposed to hypoxia. Under normoxic conditions, SA did not influence cell proliferation or LDH release of CaCo-2 cells. However, caspase-3/7 activity was significantly increased. Under hypoxia, cell proliferation was reduced and LDH release and caspase-3/7 activities were increased. None of these parameters was altered by the addition of SA under hypoxic conditions. Hypoxia increased hydrogen peroxide concentrations 300-fold and SA significantly augmented the release of hydrogen peroxide under normoxic, but not under hypoxic conditions. Phosphorylation of the pro-survival kinases akt and erk1/2 was not changed by SA under hypoxic conditions, whereas under normoxia SA reduced phosphorylation of erk1/2 after 2 hours. We conclude that in colon carcinoma cells effects of SA on apoptosis and cellular signaling are dependent on the availability of oxygen.     

Responses of type II pneumocyte antioxidant enzymes to normoxic and hyperoxic culture

Summary Cultured type II pneumocyte responses to in vitro normoxia (95% air: 5% CO₂) or hyperoxia (95% O₂:5% CO₂) were quantified. Normoxic culture (0 to 96 h) of rabbit type II cells resulted in enhanced cell-monolayer protein and DNA content. During this same time, cellular activities of superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH Px) decreased. Compared to cultures maintained in normoxia, hyperoxic exposure of cultures resulted in decreased cell-associated protein and DNA content. Exposure to hyperoxia also resulted in cytotoxicity as demonstrated by elevated cellular release of DNA, lactate dehydrogenase (LDH), and preincorporated 8-[¹⁴C]adenine. Cellular catalase and GSH Px activities in hyperoxic cells decreased similarly to normoxic controls. In contrast, cellular SOD activity in hyperoxic cells decreased less than in normoxic cultures. Cellular SOD activity in hyperoxic cultures, when normalized for cellular protein, but not DNA, was greater than normoxic values after 24 to 96 h of exposure. Unlike the decrease in cellular antioxidant enzymes during normoxic and hyperoxic culture, cellular LDH activity increased during both these exposures. Cellular LDH activity in 24 to 96 h hyperoxia-exposed cells increased to a lesser extent than normoxic controls. The extent of depression in LDH activity was dependent on whether the activity was normalized for cellular protein or DNA. Type II pneumocytes, which normally undergo hyperplasia and hypertrophy during hyperoxia in vivo, exhibited oxygen sensitivity in vitro. Exposure of type II cells to hyperoxia in vitro resulted in alterations in cellular SOD and LDH activities, but recognition of such changes were dependent on whether enzymatic activities were normalized for cellular DNA or protein. This work was supported by a grant from the Health Effects Institute, grant HL40458 from the National Institutes of Health, Bethesda, MD, and a grant from the American Lung Association, New York, NY.     

The Induction of Phagocytic Activation by Mixtures of the Water Chlorination By‐Products,Dichloroacetate‐ and Trichloroacetate,in Mice after Subchronic Exposure

In this study, groups of B6C3F1 male mice were treated with dichloroacetate (DCA), trichloroacetate (TCA), and mixtures of the compounds (Mix I, II, and III) daily by gavage, for 13 weeks. The tested doses were 7.5, 15, and 30 mg DCA/kg/day and 12.5, 25, and 50 mg TCA/kg/day. The DCA: TCA ratios in Mix I, II, and III were 7.5:12.5, 15:25, and 30:50 mg/kg/day, respectively. Peritoneal lavage cells were collected at the end of the treatment period and assayed for the biomarkers of phagocytic activation, including superoxide anion and tumor necrosis factor‐alpha production, and myeloperoxidase activity. The mixtures produced nonlinear effects on the biomarkers of phagocytic activation, with Mix I and II effects were found to be additive, but Mix III effects were found to be less than additive. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:237‐242, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21476     

Modulation of macrophage phenotype by soluble product(s) released from neutrophils

The regulation of macrophage phenotype by neutrophils was studied in the s.c. polyvinyl alcohol sponge wound model in mice made neutropenic by anti-Gr-1 Ab, as well as in cell culture. Wounds in neutropenic mice contained 100-fold fewer neutrophils than those in nonneutropenic controls 1 day after sponge implantation. Wound fluids from neutropenic mice contained 68% more TNF-alpha, 168% more IL-6, and 61% less TGF-beta1 than those from controls. Wound fluid IL-10 was not different between the two groups, and IL-4 was not detected. Intracellular TNF-alpha staining was greater in cells isolated from neutropenic wounds than in those from control wounds. The hypothesis that wound neutrophil products modulate macrophage phenotype was tested in Transwell cocultures of LPS-stimulated J774A.1 macrophages and day 1 wound cells (84% neutrophils/15% macrophages). Overnight cocultures accumulated 60% less TNF-alpha and IL-6 than cultures of J774A.1 alone. The suppression of cytokine release was mediated by a soluble factor(s), because culture supernatants from wound cells inhibited TNF-alpha and IL-6 release from LPS-stimulated J774A.1 cells. Culture supernatants from purified wound neutrophils equally suppressed TNF-alpha release from LPS-stimulated J774A.1 cells. Wound cell supernatants also suppressed TNF-alpha and superoxide release from murine peritoneal macrophages. The TNF-alpha inhibitory factor has a molecular mass <3000 Da and is neither PGE2 nor adenosine. The present findings confirm a role for neutrophils in the regulation of innate immune responses through modulation of macrophage phenotype.     

Enzymatic responses to radiation in cultured vascular endothelial and smooth muscle cells

Confluent monolayers of bovine aortic endothelial and smooth muscle cells were exposed to 0-5.0 Gy of 60Co gamma rays. From 0 to 72 hr after irradiation, the monolayer and culture medium were analyzed for cell (nuclei) number, DNA and protein content, the activities of angiotensin converting enzyme (ACE), lactate dehydrogenase (LDH), and superoxide dismutase (SOD), and LDH isoenzyme profile. Irradiated endothelial cells exhibited a time- and dose-dependent increase in cell detachment, decreased DNA and protein content and reduced ACE active per attached cell, increased LDH and SOD activities per microgram of DNA, and increased LDH activity in the culture medium. The latter was accompanied by a shift from LDH 1 to LDH 4 and 5. The release of LDH activity, observed after 0.5 Gy, was the most sensitive endothelial response, and occurred independent of or preceding cell detachment. Vascular smooth muscle cells contained two to three times more SOD activity than did endothelial cells and exhibited no significant responses to 5.0 Gy.     

Superoxide dismutase and catalase in Azotobacter vinelandii grown in continuous culture at different dissolved oxygen concentrations

Superoxide dismutase and catalase activities were studied in Azotobacter vinelandii grown diazotrophically at different ambient oxygen concentrations in continuous culture. Activities were expressed either as specific activity or activity per cell. Specific superoxide dismutase activity increased by a factor of 1.6 with increasing oxygen concentration from about 1% to 90% air saturation of the growth medium whereas specific catalase activity increased only slightly, if at all. Since cell volumes increased in parallel to increases in the oxygen concentration cellular superoxide dismutase activities increased by a factor of 4.3 while cellular catalase activities increased by a factor of 3.3. Under all conditions only the Fe-containing form of superoxide dismutase was detected. The possible function of these enzymes in the protection nitrogenase from oxygen damage is discussed.Abbreviation SOD superoxide dismutase     

Carbon monoxide inhibits superoxide dismutase and stimulates reactive oxygen species production by Desulfovibrio desulfuricans 1388

The hypothesis that oxidative stress characterized by enhanced superoxide generation underlies the toxicity of some factors to living organisms has been investigated. It is shown that CO (5-6% in gas phase) changed some growth parameters (mu, t(d)) of the sulfate-reducing bacterium Desulfovibrio desulfuricans 1388. Enhanced O(2)(-) generation registered by EPR spectroscopy and adrenochrome method was observed when cells were incubated under CO. The SOD activity in cells from the exponential growth phase growing under CO was decreased 1.5-fold compared with the control cells growing under Ar. SOD activities in cells from the stationary growth phase growing with or without CO were comparable. The results support the concept that CO toxicity for sulfate-reducing bacteria is an oxidative stress that arises in cells oxidizing CO to CO(2).     

Cells of the J774 macrophage cell line are primed for antibody-dependent cell-mediated cytotoxicity following exposure to gamma-irradiation.

Activation of macrophages (M phi) for host defense against tumor cells follows a sequence of priming events followed by an initiating stimulus that results in production and release of cytotoxic molecules that mediate target cell killing. We have developed a model to study specific macrophage cytotoxicity in vitro utilizing a cultured murine M phi cell line, J774. Specific cytotoxicity of cultured human gastrointestinal tumor cells is achieved in the presence of murine IgG2a monoclonal antibody (mAb) 17-1-A. The ability of these cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) is greatly enhanced following gamma-irradiation. ADCC can be demonstrated at mAb 17-1-A concentrations greater than or equal to 1 microgram/ml and effector/target cell ratios greater than or equal to 2. Exposure to doses greater than or equal to 10 Gy of gamma-irradiation increases ADCC threefold. Varying the duration from J774 M phi exposure to gamma-irradiation until addition of antibody-coated target cells showed that the primed state for ADCC is stable for at least 8 days but approximately 24 hr is required for complete development of the primed state. mAb-dependent target cell death begins 8 hr after addition of mAb and labeled target cells to primed effector cells and is complete by 24 hr. Incubation of unirradiated J774 M phi effector cells with recombinant murine interferon-gamma (rmIFN-gamma) also results in enhanced ADCC, but the extent of target cell killing achieved is less than that following priming by gamma-irradiation. Concomitant priming of gamma-irradiated J774 M phi with rmIFN-gamma increases the extent of ADCC. Further study of irradiated J774 cells may elucidate the molecular pathways utilized by M phi for achieving and maintaining the primed state for ADCC. Irradiated J774 cells will also provide a homogenous, stably primed cell type in which to examine the mechanism(s) of cytotoxicity employed by tumoricidal M phi.