Calorie restriction, stress and the ubiquitin-dependent pathway in mouse livers

Calorie restriction (R), the only known method to delay the aging process and extend mean and maximal lifespan, has been shown to delay the age-related decline in protein degradation. There are several proteolytic pathways. The ubiquitin- and ATP-dependent proteolytic pathway (UPP) is frequently associated with degradation of damaged abnormal and/or regulatory proteins. We examined the effect of aging and R on supernatants of livers taken from young (4.5 months) and old (23 months) Emory mice. Aging was associated with increased levels of endogenous ubiquitin conjugates, enhanced ability to form high molecular weight conjugates and ubiquitin activating (E1) and ubiquitin conjugating (E2) activity in the control (C) liver supernatants. The age-related increase in levels of endogenous ubiquitin conjugates in liver appears to be primarily due to increased E1 and E2 activities. R prevented the age-related increase in E1 and E2 activity, and thus prevented the age-related increase in levels of ubiquitin conjugates. In spite of the age-related increase in ubiquitin conjugates, no age-related changes in ubiquitin-dependent proteolytic pathway were observed in the C animals. R was associated with an enhanced ability (130%) to degrade beta-lactoglobulin by the ubiquitin-dependent proteolytic pathway in livers from 4.5-month-old animals relative to age-matched C livers. However, rates of the ubiquitin-dependent degradation of beta-lactoglobulin in the 23-month-old C and R animals were indistinguishable. There were no age- or diet-related differences in the ability to degrade another substrate, oxidized ribonuclease (RNase).     

Overexpression of human superoxide dismutase inhibits oxidation of low-density lipoprotein by endothelial cells

Oxidation of LDL in the subendothelial space has been proposed to play a key role in atherosclerosis. Endothelial cells produce superoxide anions (O2.-) and oxidize LDL in vitro; however, the role of O2.- in endothelial cell-induced LDL oxidation is unclear. Incubation of human LDL (200 microg/mL) with bovine aortic endothelial cells (BAECs) for 18 hours resulted in a 4-fold increase in LDL oxidation compared with cell-free incubation (22.5+/-1.1 versus 6.3+/-0.2 [mean+/-SEM] nmol malondialdehyde/mg LDL protein, respectively; P<0.05). Under similar conditions, incubation of LDL with porcine aortic endothelial cells resulted in a 5-fold increase in LDL oxidation. Inclusion of exogenous copper/zinc superoxide dismutase (Cu/ZnSOD, 100 microg/mL) in the medium reduced BAEC-induced LDL oxidation by 79%. To determine whether the intracellular SOD content can have a similar protective effect, BAECs were infected with adenoviral vectors containing cDNA for human Cu/ZnSOD (AdCu/ZnSOD) or manganese SOD (AdMnSOD). Adenoviral infection increased the content and activity of either Cu/ZnSOD or MnSOD in the cells and reduced cellular O2.- release by two thirds. When cells infected with AdCu/ZnSOD or AdMnSOD were incubated with LDL, formation of malondialdehyde was decreased by 77% and 32%, respectively. Two other indices of LDL oxidation, formation of conjugated dienes and increased LDL electrophoretic mobility, were similarly reduced by SOD transduction. These data suggest that production of O2.- contributes to endothelial cell-induced oxidation of LDL in vitro. Furthermore, adenovirus-mediated transfer of cDNA for human SOD, particularly Cu/ZnSOD, effectively reduces oxidation of LDL by endothelial cells.     

The effects of prenatal care utilization and maternal risk factors on pregnancy outcome between Mexican Americans and non-Hispanic whites

We have investigated, by Northern blot analysis, the hypothalamic gene expression [messenger RNA (mRNA)] of two appetite stimulating neuropeptides, neuropeptide Y (NPY) and galanin (GAL) in lean (+/+) and genetically obese (fa/fa) Zucker rats at 11, 24 and 40 weeks of age and their responsiveness to food deprivation. At 11 weeks of age, hypothalamic NPY mRNA levels of fa/fa rats were similar to those observed in lean littermates. However, NPY mRNA levels of fa/fa rats were significantly greater than those of lean rats at 24 (+126%; P < 0.01) and 40 (+65%; P < 0.05) weeks of age. Food deprivation caused a significant increase in NPY mRNA levels in both lean and fa/fa Zucker rats at 11 and 24 weeks of age, but not at 40 weeks old rats. Hypothalamic GAL mRNA showed a different pattern of change. The relative content of GAL mRNA in 11 week old obese rats was significantly lower (-68%; P < 0.05) than that of lean rats, while GAL mRNA was significantly higher in 40 week old (+57%; P < 0.05) obese rats compared to their lean littermates. At 24 weeks of age, hypothalamic GAL mRNA levels did not differ between lean and obese rats. Food deprivation induced no change in hypothalamic GAL mRNA in lean rats of all 3 ages; however, it caused an increase of GAL mRNA in obese rats at 11 (+60%; P < 0.05) and 24 (+44%; P < 0.05) weeks, but not at 40 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

(R)-alpha-lipoic acid-supplemented old rats have improved mitochondrial function, decreased oxidative damage, and increased metabolic rate

A diet supplemented with (R)-lipoic acid, a mitochondrial coenzyme, was fed to old rats to determine its efficacy in reversing the decline in metabolism seen with age. Young (3 to 5 months) and old (24 to 26 months) rats were fed an AIN-93M diet with or without (R)-lipoic acid (0.5% w/w) for 2 wk, killed, and their liver parenchymal cells were isolated. Hepatocytes from untreated old rats vs. young controls had significantly lower oxygen consumption (P<0. 03) and mitochondrial membrane potential. (R)-Lipoic acid supplementation reversed the age-related decline in O2 consumption and increased (P<0.03) mitochondrial membrane potential. Ambulatory activity, a measure of general metabolic activity, was almost threefold lower in untreated old rats vs. controls, but this decline was reversed (P<0.005) in old rats fed (R)-lipoic acid. The increase of oxidants with age, as measured by the fluorescence produced on oxidizing 2',7'-dichlorofluorescin, was significantly lowered in (R)-lipoic acid supplemented old rats (P<0.01). Malondialdehyde (MDA) levels, an indicator of lipid peroxidation, were increased fivefold with age in cells from unsupplemented rats. Feeding rats the (R)-lipoic acid diet reduced MDA levels markedly (P<0.01). Both glutathione and ascorbic acid levels declined in hepatocytes with age, but their loss was completely reversed with (R)-lipoic acid supplementation. Thus, (R)-lipoic acid supplementation improves indices of metabolic activity as well as lowers oxidative stress and damage evident in aging.     

Effect of swimming training on antioxidant enzymes in kidney of young and old mice

The current study was undertaken to investigate the effect of swimming training on the antioxidant enzyme system in kidney of young and old mice. Both young and old mice, aged 2 and 26 months old, respectively, were divided into the sedentary and swimming-trained groups. The trained mice underwent a 6-week swimming program (1 h/day, 5 days/week) in water at 35-36 degrees C. Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activity was significantly decreased with aging but was not influenced by swimming training, such changes being similar to those noted for catalase activity rather than for glutathione peroxidase activity. After swimming training Mn-SOD activity increased significantly only in old mice but was unaffected by aging. Although neither aging nor swimming training had overt effect on the expression of Cu,Zn-SOD mRNA, the immunoreactive Cu,Zn-SOD content in young mice decreased significantly after the training. Meanwhile, Mn-SOD mRNA expression in old mice was reduced by half after swimming training, accompanied by a significant decrease in its immunoreactive content; unexpectedly, however, Mn-SOD content in young mice did not parallel its mRNA expression. These findings suggest that the antioxidant enzyme system in mouse kidney trends to be down-regulated with aging, and that swimming training fails to attenuate such reduced levels of the antioxidant enzymes.     

Immunonutrition in gastric cancer surgical patients

The aim of this study was to evaluate the potential advantages of perioperative versus postoperative administration of an enteral immune-enhancing diet on host defense and protein metabolism. Thirty subjects, candidates for gastrectomy for cancer, were randomly allocated into two groups. The first group (n = 15) received an enteral formula enriched with arginine, omega-3 fatty acids, and RNA 7 d before and 7 d after surgery; the second group (n = 15) received the same diet but only 7 d after surgery. Postoperative immune and inflammatory responses were investigated by phagocytosis ability of polymorphonuclear cells, interleukin-2 receptors (IL-2R), lymphocyte subsets, interleukin-6 (IL-6), and delayed hypersensitivity response (DHR). Prealbumin (PA), retinol binding protein, albumin, and transferrin were determined as protein synthesis indicators. Perioperative immunonutrition prevented the early postoperative impairment of phagocytosis, DHR, total number of lymphocytes, and CD4/CD8 ratio (P < 0.05 versus postoperative group). The IL-2R levels were significantly higher in the perioperative group (P < 0.05 versus postoperative on postoperative day [POD] 4 and 8). Perioperative group also showed lower levels of IL-6 (P < 0.05 versus postoperative on POD 1, 4, and 8) and higher levels of PA (P = 0.04 versus postoperative on POD 8). The perioperative administration of immunonutrition ameliorated the host defense mechanisms, controlled the inflammatory response, and improved the synthesis of short half-life constitutive proteins.     

The influence of age on blood alcohol levels and ethanol-associated immunosuppression in a murine model of ethanol consumption

Several study findings indicate that with ethanol ingestion a number of changes occur in the immune system. We studied the effects of ethanol consumption on mice at various ages. We used a murine model in which young (age 6-8 weeks), middle-aged (age 12 months), and old (age 24 months) male C57Bl/6 mice were pair-fed either a Leiber-DeCarli liquid diet containing 7% (v/v) ethanol or an isocaloric control diet. Consumption of ethanol diet for 8 days resulted in high blood alcohol levels in young and old mice; low levels were observed in middle-aged mice. Middle-aged mice consumed more ethanol than did either young or old mice and had the lowest percent body weight loss of all three age groups. Proliferation of spleen lymphocytes to T-cell stimuli (concanavalin A and alloantigens) in both young and old mice fed ethanol was diminished. T-cell function was unchanged in middle-aged mice consuming an ethanol diet when compared with that observed in age-matched mice pair-fed control diet. No effect of ethanol on proliferation to lipopolysaccharide was noted in any group. Proliferative response of T cells to soluble anti-CD3 monoclonal antibody was also decreased in middle-aged and old pair-fed control mice when compared with young control mice. The proliferative response to soluble anti-CD3 in all three age groups of mice fed ethanol, however, was not significantly affected by ethanol consumption.(ABSTRACT TRUNCATED AT 250 WORDS)     

Skeletal muscle fatigue and physical endurance of young and old mice

In order to evaluate the role played by muscular and extramuscular factors in the development of fatigue in old age, the time course of fatigue in isolated skeletal muscles and spontaneous motor activity and endurance of whole animals were monitored using young (3-6 months) and old (34-36 months) CF57BL/6J mice. The isolated extensor digitorum longus (EDL) and soleus muscles from old mice had smaller (P < 0.05) mass and developed lower (P < 0.02) maximal tetanic tension at 100-Hz stimulation than the muscles of young mice. During stimulation at 30 Hz every 2.5 s, a 50% decline in original tetanic tension occurred by 109 s in young EDL and 129 s in old EDL, but by 482 s in young soleus and 1134 s (projected) in old soleus, indicating more (P < 0.05) resistance to fatigue in old than young soleus. However, the old mice showed significantly fewer (P < 0.002) spontaneous ambulatory movements than the young mice. On a treadmill with a belt speed of 10 m/min at an inclination of 0 degrees, the old mice could only run for 22 min compared to 39 min ran by young mice (P < 0.02). They took more rest periods (P< 0.02) than the young mice. In a quantitative swimming monitor, the old mice swam for a shorter (P < 0.05) time than young mice (20.4 min compared to 28.6 min). Integrated swimming activity at 20 min was smaller (P < 0.05) in old mice than in young mice (413 g/s compared to 628 g/s). Hence increased fatigue in old age is not caused by impairment of processes within the muscles, but by impairment of central or extramuscular processes.     

Venous surgery of the lower limb: value of nerve blocks

BACKGROUND: Reduction in intake of dairy products has long been recommended to reduce blood lipids. The value of monounsaturated fatty acids is increasingly recognized. METHODS: We evaluated the effects of a monounsaturate-rich butter and cheese (B) produced by modifying the bovine diet on blood lipid levels of patients with type IIa hyperlipidaemia. We compared their effects with those of normal butter and cheese (A) and polyunsaturate-rich spread and cheese (C). Using a double cross-over design, we studied 30 patients of mean age 56.4 years (23 men, one woman excluded) over 6-week periods. RESULTS: Approximately 35.5 g/day butter/cheese were consumed; no changes in serum total cholesterol, triglycerides, low-density lipoprotein, lipoprotein (a) or cholesterol: high-density lipoprotein (HDL) ratio were observed. HDL levels were higher in B(1.31 mmol/l) than in C (1.22 mmol/l; P < 0.05) and similar to those in A (1.28 mmol/l). HDL2 levels were higher in patients fed diet A(0.23 mmol/l) than they were in those fed diet C (0.19 mmol/l; P < 0.05) and similar to those in patients fed diet B (0.20 mmol/l). Serum HDL3 was significantly higher in patients fed diet B (1.11 mmol/l) than in those fed diet C (1.03 mmol/l; P < 0.05) but similar to that in patients fed diet A (1.06 mmol/l). CONCLUSIONS: Moderate intake of modified dairy products may be of value and deserves further evaluation.     

Myosin heavy chain composition in young and old rat skeletal muscle: effects of endurance exercise

The objective of this study was to determine the effects of age and exercise on the myosin heavy chain (MHC) composition of skeletal muscle. Young (3 mo) and old (22 mo) female specific pathogen-free barrier-reared Fischer 344 rats were randomly assigned to young untrained or young trained and old untrained or old trained groups, respectively. Young trained and old trained animals performed endurance exercise training on a motorized treadmill for 8 wk. Succinate dehydrogenase activity and MHC isoforms were measured in the plantaris (Plan), lateral and medial gastrocnemius (Gast), and soleus (Sol) muscles. In sedentary animals, aging resulted in a decrease (P < 0.05) in type IIb MHC and an increase (P < 0.05) in type IIa MHC in both the Gast and Plan muscles. Also, aging resulted in a small but significant increase (approximately 4%; P < 0.05) in type I MHC in the Sol. Exercise training resulted in significant (P < 0.05) increases in Gast, Plan, and Sol succinate dehydrogenase activity in both young and old animals. Furthermore, exercise training resulted in a decrease (P < 0.05) in the percentage of type IIb MHC and an increase (P < 0.05) in the percentage of type IIa MHC in the Plan in both young and old animals. These data suggest that there is an age-related shift in locomotor muscle MHC isoforms from a faster to a slower isoform.     

Alterations in the postovariectomy increases in gonadotropin secretion in middle-aged persistent-estrous rats: correlation with pituitary gonadotropin subunit gene expression

This study compared the changes in pituitary and serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) at various times following ovariectomy (OVX) between young cyclic and middle-aged persistent-estrous (PE) rats and related these to the relative gene expression of the pituitary gonadotropin subunits. In intact animals, both pituitary and serum levels of LH were similar between these two age groups, while the LH beta mRNA expression was significantly (p < 0.05) greater in young rats. Following OVX in young rats, the serum LH levels markedly increased (p < 0.05) beginning on day 7 and reaching a maximum fourfold increase by day 9. In contrast, the post-OVX increases in serum LH in middle-aged females were significantly delayed. OVX significantly (p < 0.05) increased pituitary LH contents of young rats by day 5, but had no effect on LH contents in middle-aged females until day 30 post-OVX. These changes were associated with increases in LH beta mRNA expression in both young and middle-aged females, but the levels were significantly (p < 0.05) lower in middle-aged females. Both pituitary and serum levels of FSH were significantly (p < 0.05) higher in middle-aged PE than in young rats prior to OVX, while the FSH beta mRNA expression was similar in both age groups. Following OVX in young rats, serum FSH levels rapidly increased (p < 0.05) on day 3 and attained tenfold higher values by day 30.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Coronary pressure as a determinant of B-type natriuretic peptide gene expression in isolated perfused adult rat heart

The role of coronary flow in the regulation of ventricular B-type natriuretic peptide (BNP) gene expression was studied in isolated perfused rat heart preparation. The increase of coronary flow from 5 ml/min to 20 ml/min for 2 h resulted in a 132+/-6 mm Hg increase in aortic perfusion pressure. The changes in BNP mRNA and immunoreactive BNP (IR-BNP) levels in response to hemodynamic stress were compared to those of c-fos and adrenomedullin (ADM) gene expression. The increase of coronary flow resulted in 1.5-fold increases in the left ventricular BNP mRNA (P < 0.001) and IR-BNP (P < 0.05) levels in 2-month old rats. There was also a 1.5-fold (P < 0.05) increase in ventricular c-fos mRNA levels, whereas ADM mRNA levels decreased by 74% (P < 0.001) in the left ventricle. In 18-month old rats, the increase in coronary flow decreased left and right ventricular BNP mRNA levels by 18% (P < 0.05) and 39% (P < 0.001), respectively. There were no changes in IR-BNP peptide and c-fos mRNA levels, whereas ADM mRNA levels decreased by 46% (P < 0.001) in the left ventricles. The results show that increased aortic perfusion pressure results in differential expression of cardiac genes including up-regulation of ventricular BNP and c-fos gene expression and down-regulation of ADM gene expression. Furthermore, aging seems to elevate the threshold at which hemodynamic stress of the heart results in a response at BNP gene level.     

FLP recombinase-mediated induction of Cu/Zn-superoxide dismutase transgene expression can extend the life span of adult Drosophila melanogaster flies

Yeast FLP recombinase was used in a binary transgenic system ("FLP-OUT") to allow induced overexpression of catalase and/or Cu/Zn-superoxide dismutase (Cu/ZnSOD) in adult Drosophila melanogaster. Expression of FLP recombinase was driven by the heat-inducible hsp70 promoter. Once expressed, FLP catalyzed the rearrangement and activation of a target construct in which expression of catalase or Cu/ZnSOD cDNAs was driven by the constitutive actin5C promoter. In this way a brief heat pulse (120 or 180 min, total) of young adult flies activated transgene expression for the rest of the life span. FLP-OUT allows the effects of induced transgene expression to be analyzed in control (no heat pulse) and experimental (heat pulse) populations with identical genetic backgrounds. Under the conditions used, the heat pulse itself always had neutral or slightly negative effects on the life span. Catalase overexpression significantly increased resistance to hydrogen peroxide but had neutral or slightly negative effects on the mean life span. Cu/ZnSOD overexpression extended the mean life span up to 48%. Simultaneous overexpression of catalase with Cu/ZnSOD had no added benefit, presumably due to a preexisting excess of catalase. The data suggest that oxidative damage is one rate-limiting factor for the life span of adult Drosophila. Finally, experimental manipulation of the genetic background demonstrated that the life span is affected by epistatic interactions between the transgene and allele(s) at other loci.     

Differences in weight gain in relation to race, gender, age and education in young adults: the CARDIA Study. Coronary Artery Risk Development in Young Adults

Copper/zinc (Cu/ZnSOD) and manganese (MnSOD) superoxide dismutases which catalyze the dismutation of toxic superoxide anion, O(2-)-, to O2 and H2O2, play a major role in protecting cells from toxicity of oxidative stress. However, cells overexpressing either form of the enzyme show signs of toxicity, suggesting that too much SOD may be injurious to the cell. To elucidate the possible mechanism of this cytotoxicity, the effect of SOD on DNA and RNA strand scission was studied. High purity preparations of Cu/ZnSOD and MnSOD were tested in an in vitro assay in which DNA cleavage was measured by conversion of phage phi X174 supercoiled double-stranded DNA to open circular and linear forms. Both types of SOD were able to induce DNA strand scission generating single- and double-strand breaks in a process that required oxygen and the presence of fully active enzyme. The DNA strand scission could be prevented by specific anti-SOD antibodies added directly or used for immunodepletion of SOD. Requirement for oxygen and the effect of Fe(II) and Fe(III) ions suggest that cleavage of DNA may be in part mediated by hydroxyl radicals formed in Fenton-type reactions where enzyme-bound transition metals serve as a catalyst by first being reduced by superoxide and then oxidized by H2O2. Another mechanism was probably operative in this system, since in the presence of magnesium DNA cleavage by SOD was oxygen independent and not affected by sodium cyanide. It is postulated that SOD, by having a similar structure to the active center of zinc-containing nucleases, is capable of exhibiting non-specific nuclease activity causing hydrolysis of the phosphodiester bonds of DNA and RNA. Both types of SOD were shown to effectively cleave RNA. These findings may help explain the origin of pathology of certain hereditary diseases genetically linked to Cu/ZnSOD gene.     

The effects of low dietary copper intake during pregnancy on physiological fluids and reproductive performance of first-litter gilts

Seven pairs of first littermate gilts were used to study the influence of low copper supply and pregnancy on physiological fluids and reproductive performance of first-litter gilts. They were fed semi-purified diets containing either 2.13 or 12.25 micrograms/kg of Cu from 30 days of gestation through two weeks of lactation. Low-Cu gilts had lower plasma Cu in early- and mid- gestation and farrowed piglets with lower plasma Cu and higher plasma Zn concentrations (p < 0.05). Plasma Fe and Mn concentrations were not affected by Cu supply (p > 0.05). Plasma Cu and Fe levels of newborn piglets were lower than those of their dams (p < 0.05) but this was not true for plasma Zn and Mn (p > 0.05). Low-Cu gilts had lower Cu and higher Zn content in colostrum and also lower Cu in milk than control gilts (p < 0.05). No significant differences were found in Fe and Mn levels in colostrum and milk between the two treatments (p > 0.05). Colostrum was richer in Cu and Zn than milk (p < 0.05) but not in Fe and Mn (p > 0.05). The low-Cu diet did not affect (p > 0.05) weight changes during pregnancy. The duration of parturition was shorter for low-Cu than for control gilts (3.19h vs. 5.71h, p < 0.05). Control gilts farrowed larger litters than low-Cu gilts (9.71 vs. 7.57 piglets, p < 0.05). There were no significant differences in live litter weights at birth, one wk. or two wks. of age (p > 0.05). The results indicated that a low-Cu diet and pregnancy had some effect on plasma, colostrum and milk mineral concentrations, as well as on litter size of gilts. An interaction between Cu and Zn was found.     

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.