Inhibition of cholinephosphotransferase activity in lung injury induced by 2-chloroethyl ethyl sulfide, a mustard analog

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.     

Signal transduction events in lung injury induced by 2-chloroethyl ethyl sulfide,a mustard analog

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.     

Desensitization of β‐adrenergic receptors in lung injury induced by 2‐chloroethyl ethyl sulfide,a mustard analog

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 K_D and B_max at the high‐affinity site, it significantly decreased both K_D and B_max 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     

Induction of neuronal damage in guinea pig brain by intratracheal infusion of 2-chloroethyl ethyl sulfide, a mustard gas analog

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.     

Prophylactic protection by N-acetylcysteine against the pulmonary injury induced by 2-chloroethyl ethyl sulfide,a mustard analogue

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 alpha (TNF-alpha), ceramides, and nuclear factor kappaB 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 (125)I-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-alpha, activation of neutral and acid sphingomyelinases, production of ceramides, activation of caspases, leakage of (125)I-bovine serum albumin ((125)I-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.     

Evidence of hair loss after subacute exposure to 2-chloroethyl ethyl sulfide, a mustard analog, and beneficial effects of N-acetyl cysteine

Mustard gas has been used as a vesicant chemical warfare agent. However, a suitable biomarker for monitoring mustard gas exposure is not known. We observed that the hairs of the guinea pigs exposed intratracheally to subacute doses of 2-chloroethyl ethyl sulfide (CEES), a mustard analog, came out very easily though there was no sign of skin lesions or skin damage. Also the hairs looked rough and dry and lost the shiny glaze. There was no recovery from this hair loss, though the animals never became hairless, following CEES exposure. Hairs were observed in this study both visually and with light microscopy. Treatment with N-acetylcysteine (NAC) prior to CEES exposure could prevent the hair loss completely. Hence, sudden hair loss might be a good biomarker for subacute exposure of mustard gas to subjects at risks when the victims might have no other visible symptom of toxicity.     

Decrease in brain POMC mRNA expression and onset of obesity in guinea pigs exposed to 2-chloroethyl ethyl sulfide, a mustard analogue

The full spectrum of physiological effects resulting from exposure to sulfur mustard and its analogs is currently unknown. In a guinea pig model, initially selected to study the role of an inflammatory cytokine cascade in mustard gas induced lung injury, we observed significant body weight gain in guinea pigs exposed to an intratracheally injected single dose of 2-chloroethyl ethyl sulfide, a mustard analog. The body weight gain was not associated with any apparent change in appetite. To further elucidate a molecular basis for the observed weight gain, we evaluated candidate genes for the obese phenotype by quantitative RT-PCR. We observed a time- and dose-dependent decrease in guinea pig pro-opiomelanocortin (POMC) message following treatment with mustard gas. This reduction in POMC message is consistent with the onset of obesity in the animals. We hypothesize that the POMC melanocortin pathway provides a mechanistic basis for the observed effects of sulfur mustard on body weight.     

Change of Cu,Zn-superoxide dismutase activity of guinea pig lung in experimental asthma

Correlation between the level of reactive oxygen species (ROS) generated by airway inflammatory cells and superoxide dismutase (SOD) activity of pulmonary tissue during an asthma attach was investigated in a guinea pig model of allergic asthma. In addition, the influence of SOD inhibition by diethyldithiocarbamate (DDC, Cu-chelating agent) on the airway was investigated in terms of pulmonary function during an asthma attach. Relative to controls, the capacity of bronchoalveolar lavage fluid (BAL) cells to release ROS was significantly increased in guinea pigs sensitized with ovalbumin (OA) as the antigen, and significantly increased in guinea pigs with an asthma attack provoked by the inhalation of OA. SOD activity was increased significantly in the antigen-sensitized group. The asthma provocation group showed a tendency for increase in total SOD activity, compared with the sensitization group, whose increase was dependent on the increase in copper, zinc-SOD (Cu, Zn-SOD) activity. Pretreatment with DDC increased the severity and duration of the asthma attack. These results were indicated that Cu, Zn-SOD was closely involved in the asthma process, particularly in the scavenging of oxygen radicals secreted from BAL cells.     

A spectroscopic characterization of a monomeric analog of copper,zinc superoxide dismutase

A mutated protein of human Cu(II)₂Zn(II)₂ SOD in which residues Phe50 and Gly51 at the dimer interface were substituted by Glu's, thus producing a monomeric species, has been characterized by electronic absorption spectroscopy, EPR, relaxivity and¹H NMR techniques. Such substitutions and/or accompanying remodeling and exposure of the dimer interface to solvent, alter the geometry of the active site: increases in the axiality of the copper chromophore and the Cu-OH₂ distance have been observed. The affinity of both metal binding sites for Co(II) is also altered. The observed NMR parameters of the Co(II) substituted derivative have been interpreted as a function of the decrease of rotational correlation time as a consequence of the lower molecular weight of the mutated protein. Sharper NMR signals are also obtained for the reduced diamagnetic enzyme. Results are consistent with an active site structure similar to that observed for the dimeric analog Thr137Ile characterized elsewhere. An observed proportional decrease in enzymatic activity and affinity for the N₃-anion suggests the importance of electrostatic forces during substrate docking and catalysis.     

Biosynthesis and urinary excretion of methyl sulfonium derivatives of the sulfur mustard analog, 2-chloroethyl ethyl sulfide, and other thioethers

Thioether methyltransferase was previously shown to catalyze the S-adenosylmethionine-dependent methylation of dimethyl selenide, dimethyl telluride, and various thioethers to produce the corresponding methyl onium ions. In this paper we show that the following thioethers are also substrates for this enzyme in vitro: 2-hydroxyethyl ethyl sulfide, 2-chloroethyl ethyl sulfide, thiodiglycol, t-butyl sulfide, and isopropyl sulfide. To demonstrate thioether methylation in vivo, mice were injected with [methyl-3H]methionine plus different thioethers, and extracts of lungs, livers, kidneys, and urine were analyzed by high-performance liquid chromatography for the presence of [3H]methyl sulfonium ions. The following thioethers were tested, and all were found to be methylated in vivo: dimethyl sulfide, diethyl sulfide, methyl n-propyl sulfide, tetrahydrothiophene, 2-(methylthio)ethylamine, 2-hydroxyethyl ethyl sulfide, and 2-chloroethyl ethyl sulfide. This supports our hypothesis that the physiological role of thioether methyltransferase is to methylate seleno-, telluro-, and thioethers to more water-soluble onium ions suitable for urinary excretion. Conversion of the mustard gas analog, 2-chloroethyl ethyl sulfide, to the methyl sulfonium derivative represents a newly discovered mechanism for biochemical detoxification of sulfur mustards, as this conversion blocks formation of the reactive episulfonium ion that is the ultimate alkylating agent for this class of compounds.     

Mitochondrial Cu,Zn-superoxide dismutase mediates pulmonary fibrosis by augmenting H2O2 generation

The release of H(2)O(2) from alveolar macrophages has been linked to the development of pulmonary fibrosis, but little is known about its source or mechanism of production. We found that alveolar macrophages from asbestosis patients spontaneously produce high levels of H(2)O(2) and have high expression of Cu,Zn-superoxide dismutase (SOD). Because Cu,Zn-SOD is found in the mitochondrial intermembrane space (IMS), we hypothesized that mitochondrial Cu,Zn-SOD-mediated H(2)O(2) generation contributed to pulmonary fibrosis. Asbestos-induced translocation of Cu,Zn-SOD to the IMS was unique to macrophages and dependent on functional mitochondrial respiration and the presence of at least one of the conserved cysteines required for disulfide bond formation. These conserved cysteine residues were also necessary for enzyme activation and H(2)O(2) generation. Cu,Zn-SOD-mediated H(2)O(2) generation was inhibited by knockdown of the iron-sulfur protein, Rieske, in complex III. The role of Cu,Zn-SOD was biologically relevant in that Cu,Zn-SOD(-/-) mice generated significantly less H(2)O(2) and had less oxidant stress in bronchoalveolar lavage fluid and lung parenchyma. Furthermore, Cu,Zn-SOD(-/-) mice did not develop pulmonary fibrosis, and knockdown of Cu,Zn-SOD in monocytes attenuated collagen I deposition by lung fibroblasts. Our findings demonstrate a novel mechanism for the pathogenesis of pulmonary fibrosis where the antioxidant enzyme Cu,Zn-SOD translocates to the mitochondrial IMS to increase H(2)O(2) generation in alveolar macrophages.     

Effects of niacin on bleomycin-induced increases in myeloperoxidase,prolyl hydroxylase,and superoxide dismutase activities and collagen accumulation in the lungs of hamsters

It has been shown that lung nicotinamide adenine dinucleotide (NAD) depletion accompanies bleomycin (BL)-induced lung fibrosis in the hamster and that treatment with niacin (NA), a precursor of NAD, was found to attenuate lung fibrosis caused by this agent. Niacin was used in the present study to investigate changes in some biochemical parameters and enzymes involved in the development of BL-induced lung fibrosis in the hamster. Niacin (500 mg/kg, IP), or an equivalent volume of saline (SA, IP), was given daily 2 days prior to intratracheal instillation of BL (7.5 U/5 mL/ kg) or SA and everyday thereafter throughout the study. Hamsters were killed at 1, 4, 7, 10, and 14 days after the BL or SA instillation and their lungs processed for various biochemical assays. Hydroxyproline content and superoxide dismutase (SOD) activity in SABL treated animals were significantly (P ± 0.05) elevated at 7 and 10 days, peaking at 14 days to 161 ± 11% and 159 ± 11% of the SASA treated animals, respectively. Although the hydroxyproline level of NABL treated animals was significantly elevated at 7 and 10 days and peaked at 14 days to 123 ± 8% of the NASA control, these values were significantly lower than the SABL treated animals at the corresponding times. The lung SOD activity of NABL groups was significantly higher at 4 days but significantly lower at 10 and 14 days than the SABL groups at the corresponding times. Prolyl hydroxylase (PH) activity and total lung calcium in SABL treated groups were significantly elevated compared to SASA treated groups starting at 4 days, with PH peaking at 10 days to 163 ± 13% and calcium peaking at 7 days to 148 ± 8% of SASA treated groups. The NABL treated animals displayed a significant elevation in PH activity at 4 days only (132 ± 15%), while the calcium content in this group was significantly increased at 4 and 14 days compared to NASA treated animals. However, the activity of PH in the NABL treated animals was significantly lower than the SABL treated animals at 7, 10, and 14 days. The calcium content of the NABL group was significantly lower than the SABL group at 7 and 10 days. The thiobarbituric acid reactive substance equivalents (TBARS) content and myeloperoxidase (MPO) activity were significantly elevated at all time points in SABL groups as compared to SASA groups, with peak elevation of TBARS to 160 ± 9% at 4 days and MPO to 268 ± 40% at 1 day. The TBARS content of NABL groups was significantly elevated above NASA groups at 1, 4, 7, and 10 days. The MPO activity in NABL groups was significantly elevated above NASA groups at 1, 4, 7, and 14 days, but the activity was significantly lower than SABL groups at 4 and 14 days. The data suggest that NA inhibits the extent of oxidative damage or enhances processes involved in the repair of BL-induced alveolar epithelial damage.     

Differences in mRNA expression,protein content,and enzyme activity of superoxide dismutases in type II pneumocytes of acute and chronic lung injury

The lung is protected against oxidative stress by a variety of antioxidants and type II pneumocytes seem to play an important role in antioxidant defense. Previous studies have shown that inhalation of NO₂ results in acute and chronic lung injury. How the expression and enzyme activity of antioxidant enzymes are influenced in type II cells of different inflammatory stages has yet not been studied. To elucidate this question, we exposed rats to 10 ppm NO₂ for 3 or 20 days to induce acute or chronic lung injury. From these and air-breathing rats, type II pneumocytes were isolated. The mRNA expression and protein content of CuZnSOD and MnSOD as well as total SOD-specific enzyme activity were determined. For the acute lung injury (3 d NO₂), the expression of CuZnSOD mRNA was significantly increased, while MnSOD expression was significantly reduced after 3 days of NO 2 exposure. For the chronic lung injury (20 d NO₂), CuZnSOD expression was still enhanced, while MnSOD expression was comparable to control. In parallel to CuZnSOD mRNA expression, the protein amount was significantly increased in acute and chronic lung injury however MnSOD protein content exhibited no intergroup differences. Total SOD enzyme activity showed a significant decrease after 3 days of NO₂ exposure and was similar to control after 20 days. We conclude that during acute and chronic lung injury in type II pneumocytes expression and protein synthesis of CuZnSOD and MnSOD are regulated differently.     

Mechanisms of sulfur mustard analog 2-chloroethyl ethyl sulfide-induced DNA damage in skin epidermal cells and fibroblasts

Employing mouse skin epidermal JB6 cells and dermal fibroblasts, here we examined the mechanisms of DNA damage by 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of sulfur mustard (SM). CEES exposure caused H2A.X and p53 phosphorylation as well as p53 accumulation in both cell types, starting at 1 h, that was sustained for 24 h, indicating a DNA-damaging effect of CEES, which was also confirmed and quantified by alkaline comet assay. CEES exposure also induced oxidative stress and oxidative DNA damage in both cell types, measured by an increase in mitochondrial and cellular reactive oxygen species and 8-hydroxydeoxyguanosine levels, respectively. In the studies distinguishing between oxidative and direct DNA damage, 1 h pretreatment with glutathione (GSH) or the antioxidant Trolox showed a decrease in CEES-induced oxidative stress and oxidative DNA damage. However, only GSH pretreatment decreased CEES-induced total DNA damage measured by comet assay, H2A.X and p53 phosphorylation, and total p53 levels. This was possibly due to the formation of GSH–CEES conjugates detected by LC-MS analysis. Together, our results show that CEES causes both direct and oxidative DNA damage, suggesting that to rescue SM-caused skin injuries, pleiotropic agents (or cocktails) are needed that could target multiple pathways of mustard skin toxicities.     

Protection of half sulfur mustard gas–induced lung injury in guinea pigs by antioxidant liposomes

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 (α, γ, δ), N‐acetylcysteine (NAC), and glutathione (GSH). A single dose (200 µL) 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 α‐tocopherol, 11 mM γ‐tocopherol, and 75 mM NAC. However, LIP‐2 contained additionally 5 mM δ‐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. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:143–153, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20279     

Oxidation of Cu,Zn-superoxide dismutase by the myeloperoxidase/hydrogen peroxide/chloride system: functional and structural effects

This study investigated the functional and structural effects of bovine Cu,Zn-superoxide dismutase (Cu,Zn-SOD) oxidation by the myeloperoxidase (MPO)/hydrogen peroxide (H 2 O 2 )/chloride system and reagent hypochlorous acid (HOCl). Exposure to HOCl led to a fast inactivation accompanied by structural alterations. The residual SOD activity depended on the reactants concentration ratio and on the exposure time. The concomitant high consumption of HOCl indicated the presence of multiple targets on the protein. As assessed by SDS/PAGE, HOCl caused the dissociation of the protein into protomers at 16 kDa stable to both SDS and reducing conditions. Results from isoelectric focusing gels showed that exposure to HOCl induced the formation of modified protein derivatives, with a more acidic net electric charge than the parent molecule, consistent with the presence of additional ions observed in the electrospray ionization mass spectra. The reaction of protein with HOCl resulted in changes in protein conformation as assessed by the UV fluorescence and oxidation of the unique methionine and tyrosine, chlorination of several lysines with formation of chloramines. There was no significant formation of dityrosine and carbonyl groups. Exposure to high levels of HOCl resulted in complete enzyme inactivation, loss of additional lysine, histidine and arginine residues and coincident detection of weakly bound zinc and copper using 4-pyridylazaresorcinol. Collectively, the results suggest that the decrease of the dismutase activity is probably related to both dissociation into protomers and unfolding due to extensive oxidative modifications of amino acids.     

Novel effect of aromatic compounds on the iron-dependent expression of theEscherichia coli K12 manganese superoxide dismutase (sodA) gene

Summary InEscherichia coli, the superoxide dismutase genes (sodA andsodB) sense the availability of Fe through the action of thefur locus [E. C. Niederhoffer, C. M. Naranjo, K. L. Bradley, J. A. Fee (1990) Control ofEscherichia coli superoxide dismutases (sodA andsodB) genes by the ferric uptake regulation (fur) locus,J. Bacteriol. 172, 1930–1938]. Previous work from other laboratories has shown that a variety of metal chelators and of redox-active aromatic compounds can dramatically induce expression ofsodA. Here we show that non-redox-active, non-metal-chelating aromatic compounds also enhance expression of a chromosomalsodA gene fusion and that these effects are strongly modulated by the Fur phenotype (Fur^±) and by the availability of iron in the culture medium. The compounds studied were ethidium bromide, hemin, 2,2-bipyridine, 1,10-phenantroline, 4,7-phenantroline, rhodamine B1, rhodamine 6G, and, for comparison to previous studies, Paraquat.Abbreviations DTPA diethylenetriaminepentaacetic acid - Paraquat N,N-dimethyl-1,1-bipyridene - bpy 2,2-bipyridine - phen 1,10-phenanthroline - 4,7-phen 4,7-phenanthroline     

Cloning of a superoxide dismutase gene from Listeria ivanovii by functional complementation in Escherichia coli and characterization of the gene product.

Summary A gene encoding superoxide dismutase (EC 1.15.1.1., SOD) was isolated from a plasmid library of chromosomal DNA from Listeria ivanovii by functional complementation of an SOD-negative Escherichia coli host. The nucleotide sequence of the cloned gene was determined and contained an open reading frame which codes for a protein of 202 amino acid residues (calculated molecular weight 22 755 Da including the amino-terminal methionine residue). Comparison of the deduced amino acid sequence of L. ivanovii SOD with previously reported SOD amino acid sequences revealed considerable homologies with Fe- and Mn-dependent SODs. Enzymatic analyses using cell lysates and the purified recombinant enzyme indicated that this SOD is manganese-dependent. The recombinant SOD accounted for up to 30% of the total soluble protein in recombinant E. coli and protected sodA sodB mutants against the toxic effects of paraquat. Subunits of the recombinant Listeria SOD and of both E. coli SODS formed enzymatically active hybrids in vivo.Some of our preliminary observations have been published as a conference report of SOD V (Jerusalem, 1989) in Free Rad Res Commun (1991) 12–13:371