Potato mitochondrial manganese superoxide dismutase is an RNA-binding protein

We have investigated the toxicity of the cholesterol oxidation products (oxysterols), 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, 7-ketocholesterol, 25-hydroxycholesterol and 26-hydroxycholesterol to human monocyte-macrophages in vitro. The 7-position derivatives are present in low density lipoprotein (LDL) oxidised with copper (II) sulphate and macrophages, and in extracts of human atherosclerotic lesions, which also contain 26-hydroxycholesterol. We have also assessed 25-hydroxycholesterol for toxicity because it has often been used in studies of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibition and LDL receptor down-regulation. Measurement of radioactivity release from monocyte-macrophages preloaded with tritiated adenine, as a means of assessing cytotoxicity that all the oxysterols showed time- and concentration-dependent toxicity. The cytotoxic potency of 26-hydroxycholesterol was the greatest. The 7-position derivatives also produced marked cell damage, though at higher concentrations than for 26-hydroxycholesterol. Of the oxysterols assessed, the toxicity of 25-hydroxycholesterol was the least. The cytotoxicity of 7 beta-hydroxycholesterol and 26-hydroxycholesterol was also shown using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye reduction assay which confirmed that 26-hydroxycholesterol was more toxic than 7 beta-hydroxycholesterol. Incubation of monocyte-macrophages with cholesterol added to the different oxysterols gave varying results. Cholesterol, which was not itself toxic, inhibited the toxicity of 25-hydroxycholesterol and 26-hydroxycholesterol, but the toxicity of the 7-position derivatives was not affected. The possible relevance of these molecules to the death of macrophages seen in atherosclerosis is discussed.     

Ovarian function in superoxide dismutase 1 and 2 knockout mice

Copper/zinc superoxide dismutase (SOD1) and manganese superoxide dismutase (SOD2) are the two major intracellular enzymes which inactivate superoxide radicals. SOD1 is present in both cytoplasmic and nuclear compartments whereas SOD2 is localized to mitochondria. Both enzymes are expressed in multiple tissues as well as ovaries of several species including humans and rodents. Dominant mutations in SOD1 are associated with amyotrophic lateral sclerosis. We have previously demonstrated that SOD2-deficient mice die within three weeks of birth due to oxidative mitochondrial injury in central nervous system neurons and cardiac myocytes. In this report, we demonstrate that female homozygous mutant mice lacking SOD1 can survive to the adult stage but are subfertile. Whereas breeding of 5 SOD1 heterozygote females produced an average of 1.0 litter/month with 8.6 offspring/litter (n = 31 litters), only 11 of 16 SOD1 homozygote mice over a 2-6 month period became pregnant averaging 0.23 litters/month with an average litter size of 2.7 (n = 21 litters). Histological analysis of the ovaries from SOD1-deficient mice often reveals many primary and small antral follicles but few corpora lutea. In addition, ovaries from postnatal SOD2-deficient mice, transplanted to the bursa of wild-type hosts, show all stages of folliculogenesis including corpora lutea and can give rise to viable offspring. These studies support an important role of SOD1 in female reproductive function and suggest that SOD2 is not essential for ovarian function.     

Increased 3-nitrotyrosine and oxidative damage in mice with a human copper/zinc superoxide dismutase mutation

The structural and functional barrier preventing the free advancement of microbial plaque subgingivally along the tooth surface is formed by the junctional epithelial (JE) cells directly attached to the tooth (DAT cells). The mechanism leading to degeneration of the DAT cells is not known. In the present study we examined the possible role of short chain fatty acids (SCFAs) on epithelial cells by making use of 2 epithelial cell cultures (HaCaT and ERM) and an explant culture model of human JE. The SCFAs butyrate and propionate were used in concentrations found in human plaque and gingival crevicular fluid (0.25-16.0 mM). The SCFAs had no effect on primary cell adhesion nor on the epithelial attachment apparatus (EAA). By contrast, even 0.25 mM of butyrate significantly retarded epithelial cell growth. Similar effects with propionate were first observed at concentrations higher than 1.0 mM. The retardation of epithelial cell growth was found to be due to inhibition of cell division. Furthermore, after butyrate treatment dense accumulations of intermediate filaments and cytoplasmic vacuolization were characteristically seen in cells adjacent to cells of normal appearance. This suggests that some cells of the growing epithelial cell population are more sensitive to the SCFAs than others, and agrees with previous reports on the DAT cells of periodontally-involved teeth in vivo. The results suggest that SCFAs are microbial factors that play a role in the initiation and progression of periodontal pocket formation by impairing epithelial cell function rather than having a direct effect on the EAA.     

Kainate-induced hippocampal DNA damage is attenuated in superoxide dismutase transgenic mice

Peripheral administration of kainic acid (KA) can cause cell death in the hippocampus of rodents. This is thought to involve oxidative stress. In the present study, we tested the possibility that KA-induced neuronal cell death might be attenuated in CuZn superoxide dismutase transgenic (SOD-Tg) mice. Acute administration of KA causes animal death in a dose-dependent fashion; this was attenuated in SOD-Tg mice. Similarly, KA caused dose-dependent neuronal cell death in the hippocampus of wild-type mice; this cell death was attenuated in the SOD-Tg mice, in a gene-dosage-dependent fashion, with homozygous mice showing complete protection even at the highest dose (45 mg/kg) of KA used in this study. These results provide further support for the involvement of oxygen-based radicals in the toxic effects of KA.     

Mitochondrial susceptibility to oxidative stress exacerbates cerebral infarction that follows permanent focal cerebral ischemia in mutant mice with manganese superoxide dismutase deficiency

Since 1989, about 570 different p53 mutations have been identified in more than 8000 human cancers. A database of these mutations was initiated by M. Hollstein and C. C. Harris in 1990. This database originally consisted of a list of somatic point mutations in the p 53 gene of human tumors and cell lines, compiled from the published literature and made available in a standard electronic form. The database is maintained at the International Agency for Research on Cancer (IARC) and updated versions are released twice a year (January and July). The current version (July 1997) contains records on 6800 published mutations and will surpass the 8000 mark in the January 1998 release. The database now contains information on somatic and germline mutations in a new format to facilitate data retrieval. In addition, new tools are constructed to improve data analysis, such as a Mutation Viewer Java applet developed at the European Bioinformatics Institute (EBI) to visualise the location and impact of mutations on p53 protein structure. The database is available in different electronic formats at IARC (http://www.iarc. fr/p53/homepage.htm ) or from the EBI server (http://www.ebi.ac.uk ). The IARC p53 website also provides reports on database analysis and links with other p53 sites as well as with related databases. In this report, we describe the criteria for inclusion of data, the revised format and the new visualisation tools. We also briefly discuss the relevance of p 53 mutations to clinical and biological questions.     

Inhibitory effects of recombinant manganese superoxide dismutase on influenza virus infections in mice

The oxygen free-radical scavenger recombinant human manganese superoxide dismutase (MnSOD) was studied for its effects on influenza virus infections in mice when used alone and in combination with ribavirin. Mice challenged with influenza A/NWS/33 (H1N1) virus were treated parenterally in doses of 25, 50, and 100 mg/kg of body weight per day every 8 h for 5 days beginning at 48 h post-virus exposure. An increase in mean day to death, lessened decline in arterial oxygen saturation, and reduced lung consolidation and lung virus titers occurred in the treated animals. To determine the influence of viral challenge, experiments were run in which mice were infected with a 100 or 75% lethal dose of virus and were treated intravenously once daily for 5 days beginning 96 h after virus exposure. Weak inhibition of the mortality rate was seen in mice receiving the high viral challenge, whereas significant inhibition occurred in the animals infected with the lower viral challenge, indicating that MnSOD effects are virus dose dependent. To determine if treatment with small-particle aerosol would render an antiviral effect, infected mice were treated by this route for 1 h daily for 5 days beginning 72 h after virus exposure. A dose-responsive disease inhibition was seen. An infection induced by influenza B/Hong Kong/5/72 virus in mice was mildly inhibited by intravenous MnSOD treatment as seen by increased mean day to death, lessened arterial oxygen saturation decline, and lowered lung consolidation. MnSOD was well tolerated in all experiments. A combination of MnSOD and ribavirin, each administered with small-particle aerosol, resulted in a generally mild improvement of the disease induced by the influenza A virus compared with use of either material alone.     

Role of manganese superoxide dismutase in a mucoid isolate of Pseudomonas aeruginosa: adaptation to oxidative stress

Chronic infection by alginate-producing (mucoid) Pseudomonas aeruginosa is a leading cause of morbidity among cystic fibrosis (CF) patients. In the lungs of CF patients, the bacteria are exposed to activated oxygen species produced by the phagocytes of the host or resulting from the metabolism of oxygen. Two isoforms of superoxide dismutase are synthesized by P. aeruginosa; they differ by the metal present at their active site, which is either iron or manganese. To evaluate the role of manganese-containing superoxide dismutase (MnSOD), encoded by sodA, we have isolated a sodA mutant of the mucoid P. aeruginosa strain CHA isolated from the bronchopulmonary tract of a CF patient. The sodA mutant exhibited an increased sensitivity to oxidative stress generated by paraquat and was less resistant to oxidative stress in the stationary phase of growth compared with its parental strain. It was observed that MnSOD was expressed in the parental strain solely during the stationary phase of growth and that cells of the sodA mutant taken at the stationary phase resumed growth with a longer delay than the sodA+ cells when reinoculated in a new medium, especially in the presence of paraquat. These results suggest that MnSOD may participate in the adaptation of mucoid strains of P. aeruginosa to the stationary phase of growth in the lungs of CF patients.     

Increased mitochondrial superoxide dismutase activity in Parkinson's disease but not amyotrophic lateral sclerosis motor cortex

Oxidative stress may contribute to the neurodegenerative process in amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Motor cortex in PD is not affected and its inclusion in studies of free radical involvement in ALS pathogenesis could help elucidate whether oxidative stress is disease specific or a more widespread phenomenon present in the neurodegeneration. We have measured cytosolic and mitochondrial isoforms of superoxide dismutase (SOD), antioxidant enzymes involved in primary defence against free radical damage, in motor cortex of six patients with sporadic form of ALS (SALS), eight with PD and eight normal control subjects. We have found no difference in the activities of cytosolic and mitochondrial SOD between SALS and control motor cortex. Mitochondrial SOD activity in PD motor cortex was, however, significantly higher than in SALS and control motor cortex whereas activity of cytosolic SOD was lower than in two other groups although the differences were not statistically significant. Our findings indicate the presence of an altered antioxidant defence system in PD but not ALS upper motor neurons, suggesting that oxidative stress may be a widespread phenomenon in PD.     

Prevention of mitochondrial injury by manganese superoxide dismutase reveals a primary mechanism for alkaline-induced cell death

Alkalosis is a clinical complication resulting from various pathological and physiological conditions. Although it is well established that reducing the cellular proton concentration is lethal, the mechanism leading to cell death is unknown. Mitochondrial respiration generates a proton gradient and superoxide radicals, suggesting a possible link between oxidative stress, mitochondrial integrity, and alkaline-induced cell death. Manganese superoxide dismutase removes superoxide radicals in mitochondria, and thus protects mitochondria from oxidative injury. Cells cultured under alkaline conditions were found to exhibit elevated levels of mitochondrial membrane potential, reactive oxygen species, and calcium which was accompanied by mitochondrial damage, DNA fragmentation, and cell death. Overexpression of manganese superoxide dismutase reduced the levels of intracellular reactive oxygen species and calcium, restored mitochondrial transmembrane potential, and prevented cell death. The results suggest that mitochondria are the primary target for alkaline-induced cell death and that free radical generation is an important and early event conveying cell death signals under alkaline conditions.     

A glutamate bridge is essential for dimer stability and metal selectivity in manganese superoxide dismutase

In Escherichia coli manganese superoxide dismutase (MnSOD), the absolutely conserved Glu170 of one monomer is hydrogen-bonded to the Mn ligand His171 of the other monomer, forming a double bridge at the dimer interface. Point mutation of Glu170 --> Ala destabilizes the dimer structure, and the mutant protein occurs as a mixture of dimer and monomer species. The purified E170A MnSOD contains exclusively Fe and is devoid of superoxide dismutase activity. E170A Fe2-MnSOD closely resembles authentic FeSOD in terms of spectroscopic properties, anion interactions and pH titration behavior. Reconstitution of E170A Fe2-MnSOD with Mn(II) salts does not restore superoxide dismutase activity despite the spectroscopic similarity between E170A Mn2-MnSOD and wild type Mn2-MnSOD. Growth of sodA+ and sodA- E. coli containing the mutant plasmid pDT1-5(E170A) is impaired, suggesting that expression of mutant protein is toxic to the host cells.     

Expression of manganese superoxide dismutase in esophageal and gastric cancers

The tumor-killing activity of radiotherapy and chemotherapy for cancer is closely associated with the production of active oxygen, and the relation between therapeutic resistance and active oxygen scavengers produced by the tumor itself is gaining more attention. It is considered that manganese superoxide dismutase (MnSOD) protects host cells from oxidative stress, in synergy with other antioxidant enzymes. In this study, we used a quantitative polymerase chain reaction assay to measure MnSOD mRNA in resected specimens from patients with esophageal and gastric cancers. In both esophageal and gastric cancers, the level of MnSOD mRNA was significantly elevated in cancer tissue compared to non-cancer tissue (P < 0.01). In gastric cancer tissue, the MnSOD mRNA level was significantly higher than in esophageal cancer tissue (P < 0.01). The significance of MnSOD in cancer tissue was investigated further by measuring MnSOD content in resected specimens using an enzyme-linked immunosorbent assay, and by examining its location by an immunohistochemical method. Upregulation of MnSOD in cancer tissue most likely serves as a protective mechanism against anti-cancer therapies known to produce superoxide radicals as a key component of their tumor-killing activity.     

Induction of manganese superoxide dismutase in BV-2 microglial cells

The regulation of the manganese-dependent superoxide dismutase (Mn-SOD) was studied in immortalized microglial cells (line BV-2). BV-2 cells, activated with lipopolysaccharide (LPS), exhibited an increase in Mn-SOD-like immunoreactivity, that was associated with an accumulation of nitrite in the culture medium and an increase in immunoreactivity for the inducible type of nitric oxide synthase (i-NOS). The i-NOS inhibitor L-N6-(1-iminoethyl)-lysine (NIL, 600 microM) suppressed the nitrite accumulation and the increase in Mn-SOD-like immunoreactivity in activated cells without significant effect on the level of i-NOS-like immunoreactivity. The NO donor sodium nitroprusside dose-dependently increased Mn-SOD-like immunoreactivity in NIL-pretreated BV-2 cells. These results indicate that the induction of Mn-SOD in activated BV-2 cells is mediated in part by NO, or its metabolites.     

Human mitochondrial manganese superoxide dismutase polymorphic variant Ile58Thr reduces activity by destabilizing the tetrameric interface

Human manganese superoxide dismutase (MnSOD) is a homotetrameric enzyme which protects mitochondria against oxygen-mediated free radical damage. Within each subunit, both the N-terminal helical hairpin and C-terminal alpha/beta domains contribute ligands to the catalytic manganese site. Two identical four-helix bundles, symmetrically assembled from the N-terminal helical hairpins, form a novel tetrameric interface that stabilizes the active sites. The 2.5 A crystallographic structure of the naturally occurring polymorphic variant Ile58Thr MnSOD reveals that the helical hairpin mutation Thr58 causes two packing defects in each of the two four-helix bundles of the tetrameric interface. Similar mutations, expected to cause packing defects in the Cu,ZnSOD dimer interface, are associated with the degenerative disease amyotrophic lateral sclerosis. Ile58Thr MnSOD is primarily dimeric in solution and is significantly less thermostable than the normal enzyme, with decreases of 15 degrees C in the main melting temperature and 20 degrees C in the heat-inactivation temperature. Consequently, this mutant MnSOD is compromised at normal body temperatures: thermal inactivation, predicted from the decrease in thermal stability, occurs with a theoretical half-life of only 3.2 h at 37 degrees C (1.4 h at 41 degrees C), compared with 3.1 years for native MnSOD. This prediction is supported by direct measurements: incubation at 41.7 degrees C for 3 h has no effect on the activity of native MnSOD but completely inactivates mutant MnSOD. Rapid inactivation of Ile58Thr MnSOD at the elevated temperatures associated with fever and inflammation could provide an early advantage by killing infected cells, but also would increase superoxide-mediated oxidative damage and perhaps contribute to late-onset diseases.     

Mitochondrial disease in superoxide dismutase 2 mutant mice

Oxidative stress has been implicated in many diseases. The chief source of reactive oxygen species within the cell is the mitochondrion. We have characterized a variety of the biochemical and metabolic effects of inactivation of the mouse gene for the mitochondrial superoxide dismutase (CD1-Sod2(tm1Cje)). The Sod2 mutant mice exhibit a tissue-specific inhibition of the respiratory chain enzymes NADH-dehydrogenase (complex I) and succinate dehydrogenase (complex II), inactivation of the tricarboxylic acid cycle enzyme aconitase, development of a urine organic aciduria in conjunction with a partial defect in 3-hydroxy-3-methylglutaryl-CoA lyase, and accumulation of oxidative DNA damage. These results indicate that the increase in mitochondrial reactive oxygen species can result in biochemical aberrations with features reminiscent of mitochondrial myopathy, Friedreich ataxia, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.     

Appetitively motivated instrumental learning in SynGAP heterozygous knockout mice.

The synaptic Ras/Rap-GTPase-activating protein (SynGAP) regulates specific intracellular events following N-methyl-d-aspartate receptor (NMDAR) activation. Here, the impact of SynGAP heterozygous knockout (SG+/?) on NMDAR-dependent functions was assessed using different positive reinforcement schedules in instrumental conditioning. The knockout did not affect the temporal control of operant responding under a fixed interval (FI) schedule, but led to a putative enhancement in response vigor and/or disinhibition. When examined on differential reinforcement of low rates of response (DRL) schedules, SG+/? mice showed increased responding under DRL-4s and DRL-8s, without impairing the response efficiency (total rewards/total lever presses) because both rewarded and nonrewarded presses were elevated. Motivation was unaffected as evaluated using a progressive ratio (PR) schedule. Yet, SG+/? mice persisted in responding during extinction at the end of PR training, although an equivalent phenotype was not evident in extinction learning following FI-20s training. This extinction phenotype is therefore schedule-specific and cannot be generalized to Pavlovian conditioning. In conclusion, constitutive SynGAP reduction increases vigor in the execution of learned operant behavior without compromising its temporal control, yielding effects readily distinguishable from NMDAR blockade. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Temporal variation in hepatic superoxide dismutase activity in mice

Intestinal ischemia is a common clinical event and reperfusion results in further tissue damage exceeding that of ischemia alone. The present study was designed to test this and to assess the role of pentoxifylline, (administered intravenously as a bolus dose of 25 mg/kg in 1 ml normal saline, followed by continuous infusion of 0.2 mg/kg/minute for 95 minutes), in ischemia-reperfusion injury of the rat intestine. Intestinal ischemia was produced by occlusion of the superior mesenteric artery (SMA) with interruption of the collateral flow for 30 minutes. Reperfusion was established by declamping the (SMA) for 1 hour and evaluation of the mucosal damage was determined using a grading scale from 0 to 5, with estimation of mean mucosal thickness, villous height and crypt depth. The grade of mucosal damage, mucosal thickness, villous height and crypt depth were 2.2, 407 microns, 210 microns, and 196 microns respectively in the ischemia group, and 3.6, 327 microns, 156 microns, and 171 microns respectively in the ischemia reperfusion group, while these values in ischemia reperfusion with administration of pentoxifylline group were 2.5, 505 microns, 294 microns, and 200 microns respectively. The severity of the tissue injury increased considerably after reperfusion of the ischemic intestine and pentoxifylline was effective in attenuating the reperfusion injury significantly.     

Mutagenesis of a proton linkage pathway in Escherichia coli manganese superoxide dismutase

Mutagenesis of Escherichia coli manganese superoxide dismutase (MnSD) demonstrates involvement of the strictly conserved gateway tyrosine (Y34) in exogenous ligand interactions. Conservative replacement of this residue by phenylalanine (Y34F) affects the pH sensitivity of the active-site metal ion and perturbs ligand binding, stabilizing a temperature-independent six-coordinate azide complex. Mutant complexes characterized by optical and electron paramagnetic resonance (EPR) spectroscopy are distinct from the corresponding wild-type forms and the anion affinities are altered, consistent with modified basicity of the metal ligands. However, dismutase activity is only slightly reduced by mutagenesis, implying that tyrosine-34 is not essential for catalysis and may function indirectly as a proton donor for turnover, coupled to a protonation cycle of the metal ligands. In vivo substitution of Fe for Mn in the MnSD wild-type and mutant proteins leads to increased affinity for azide and altered active-site properties, shifting the pH-dependent transition of the active site from 9.7 (Mn) to 6.4 (Fe) for wt enzyme. This pH-coupled transition shifts once more to a higher effective pKa for Y34F Fe2-MnSD, allowing the mutant to be catalytically active well into the physiological pH range and decreasing the metal selectivity of the enzyme. Peroxide sensitivities of the Fe complexes are distinct for the wild-type and mutant proteins, indicating a role for Y34 in peroxide interactions. These results provide evidence for a conserved peroxide-protonation linkage pathway in superoxide dismutases, analogous to the proton relay chains of peroxidases, and suggests that the selectivity of Mn and Fe superoxide dismutases is determined by proton coupling with metal ligands.     

Crystal structure of Y34F mutant human mitochondrial manganese superoxide dismutase and the functional role of tyrosine 34

Tyrosine 34 is a prominent and conserved residue in the active site of the manganese superoxide dismutases in organisms from bacteria to man. We have prepared the mutant containing the replacement Tyr 34 --> Phe (Y34F) in human manganese superoxide dismutase (hMnSOD) and crystallized it in two different crystal forms, orthorhombic and hexagonal. Crystal structures of hMnSOD Y34F have been solved to 1.9 A resolution in a hexagonal crystal form, denoted as Y34Fhex, and to 2.2 A resolution in an orthorhombic crystal form, denoted as Y34Fortho. Both crystal forms give structures that are closely superimposable with that of wild-type hMnSOD, with the phenyl rings of Tyr 34 in the wild type and Phe 34 in the mutant very similar in orientation. Therefore, in Y34F, a hydrogen-bonded relay that links the metal-bound hydroxyl to ordered solvent (Mn-OH to Gln 143 to Tyr 34 to H2O to His 30) is broken. Surprisingly, the loss of the Tyr 34 hydrogen bonds resulted in large increases in stability (measured by Tm), suggesting that the Tyr 34 hydroxyl does not play a role in stabilizing active-site architecture. The functional role of the side chain hydroxyl of Tyr 34 can be evaluated by comparison of the Y34F mutant with the wild-type hMnSOD. Both wild-type and Y34F had kcat/Km near 10(9) M-1 s-1, close to diffusion-controlled; however, Y34F showed kcat for maximal catalysis smaller by 10-fold than the wild type. In addition, the mutant Y34F was more susceptible to product inhibition by peroxide than the wild-type enzyme. This activity profile and the breaking of the hydrogen-bonding chain at the active site caused by the replacement Tyr 34 --> Phe suggest that Tyr 34 is a proton donor for O2* - reduction to H2O2 or is involved indirectly by orienting solvent or other residues for proton transfer. Up to 100 mM buffers in solution failed to enhance catalysis by either Y34F or the wild-type hMnSOD, suggesting that protonation from solution cannot enhance the release of the inhibiting bound peroxide ion, likely reflecting the enclosure of the active site by conserved residues as shown by the X-ray structures. The increased thermostability of the mutant Y34F and equal diffusion-controlled activity of Y34F and wild-type enzymes with normal superoxide levels suggest that evolutionary conservation of active-site residues in metalloenzymes reflects constraints from extreme rather than average cellular conditions. This new hypothesis that extreme rather than normal substrate concentrations are a powerful constraint on residue conservation may apply most strongly to enzyme defenses where the ability to meet extreme conditions directly affects cell survival.     

Induction of manganese superoxide dismutase gene expression in bronchoepithelial cells after rockwool exposure

Superoxide dismutases play an important protective role in the lung defense against the pro-oxidative effect of fibrous dusts (e.g. crocidolite fibers). Particularly crocidolite, but also other asbestos fibers, are known to induce cellular antioxidant defense. Although rockwool, a man-made fiber made from rock, is used widely for insulation purposes, its effects on the superoxide dismutases in bronchoepithelial cells have not been investigated. Thus, the purpose of this study was to determine whether human bronchoepithelial cells (BEAS 2B) respond to rockwool fibers (115-4 experimental rockwool fiber) by induction of MnSOD mRNA and an increase of MnSOD activity levels. The results were compared with BEAS 2B cells exposed to silica (alpha-quartz: DQ12; SiO2) and UICC (Union Internationale Contre le Cancer) crocidolite (concentrations of all dusts: 0, 2, 5, 10, 25, 50 micrograms/cm2 = 0, 2.4, 6, 12, 30, 60 micrograms/ml; 24-h exposure) as control fibers. Scanning electron microscopy confirmed close dust cell contact under all experimental settings. Very low MnSOD mRNA baseline levels rose significantly (p < 0.001) in BEAS 2B cells exposed to all three dusts at 2 micrograms/cm2. However, at > 25 micrograms/cm2 MnSOD mRNA levels in silica- and crocidolite- but not in rockwool-exposed cells decreased. Slight (no significance) increases of MnSOD activity were observed which decreased at higher dust (> 5 micrograms/cm2) concentrations. These results suggest that: (1) like crocidolite and silica, rockwool accelerates MnSOD gene expression in bronchoepithelial cells; (2) an increase of MnSOD mRNA levels is not accompanied by MnSOD activity elevation; (3) in contrast to rockwool, high concentrations (> or = 25 micrograms/cm2) of crocidolite and silica reduced MnSOD activity and MnSOD mRNA levels. Because oxidants (H2O2) and crocidolite fibers were shown to reduce SOD activity, lack of active MnSOD protein may be caused by inactivation on a post-translational level. Furthermore, the decline of MnSOD mRNA and MnSOD activity levels coincides with increasing cytotoxicity. In conclusion, rockwool was demonstrated to induce MnSOD gene expression, perhaps because of its pro-oxidative effect in bronchoepithelial cells. In contrast to crocidolite and silica, rockwool fibers are not cytotoxic in this experimental setting.