pp a-a February 2003 Oxygen/Nitrogen Radicals: Cell Injury and Disease
pp 1-5 February 2003
Diabetes is known to involve oxidative stress and changes in lipid metabolism. Many secondary plant metabolites have been shown to possess antioxidant activities, improving the effects of oxidative stress on diabetes. This study evaluated the effects of extracts fromGongronema latifolium leaves on antioxidant enzymes and lipid profile in a rat model of non insulin dependent diabetes mellitus (NIDDM). The results confirmed that the untreated diabetic rats were subjected to oxidative stress as indicated by significantly abnormal activities of their scavenging enzymes (low superoxide dismutase and glutathione peroxide activities), compared to treated diabetic rats, and in the extent of lipid peroxidation (high malondialdehyde levels) present in the hepatocytes. The ethanolic extract ofG. latifolium leaves possessed antioxidant activity as shown by increased superoxide dismutase and glutathione peroxidase activities and decreases in malondialdehyde levels. High levels of triglycerides and total cholesterol, which are typical of the diabetic condition, were also found in our rat models of diabetes. The ethanolic extract also significantly decreased triglyceride levels and normalized total cholesterol concentration.
pp 7-11 February 2003
Oxidative stress has been recognized as a central feature of smoke induced chronic obstructive pulmonary disease (COPD). Imbalance between oxidant and antioxidant enzymes is also an established fact in these patients. But studies in regard to stable COPD patients and effect of vitamin E supplementation are lacking. Thirty patients with COPD were included in the study. Their baseline clinical examination, spirometry, plasma malondialdehyde (MDA), alpha-tocopherol and red blood cell superoxide dismutase (SOD) levels were measured. Twenty healthy non-smokers who were matched for age and sex served as controls. All the above parameters were repeated after 12 weeks of supplementation with 400IU of vitamin E daily. The mean malondialdehyde levels in the patients at baseline were higher than controls (5-91 ±1.23 nmol/ml vs 4.55 ± 1.51 nmol/ml,P = 0.001), so also was plasma alpha-tocopherol levels (P<0.00l), while SOD levels were lower in the patients compared to controls (1692 ± 259 units g/Hb vs 2451 ± 131 units g/Hb,P < 0001). Exogenous vitamin E (400 IU per day) supplementation did not bring about any significant change in plasma alpha-tocopherol and SOD levels. The Pearson’s co-efficient of correlation between the levels of MDA, vitamin E, SOD; and spirometric measurements were not significant either on day 1 or after 12 weeks of vitamin E supplementation. The present study shows that initially the plasma lipid peroxide (MDA) levels are high and antioxidants (alpha-tocopherol and SOD) are low in patients with COPD. Exogenous supplementation with vitamin E does not have any significant effect on the spirometric measurements though it brings down the levels of MDA showing attenuation of further damage. However, inclusion of larger number of patients and supplementation with vitamin E for longer periods may throw more light on free radical injury and protective effects of antioxidants.
pp 13-18 February 2003
Inhalation of residual oil fly ash (ROFA) increases pulmonary morbidity in exposed workers. We examined the role of reactive oxygen species (ROS) in ROFA-induced lung injury. ROFA was collected from a precipitator at Boston Edison Co., Everett, MA, USA. ROFA (ROFA-total) was suspended in saline, incubated for 24 h at 37°C, centrifuged, and separated into its soluble (ROFA-sol.) and insoluble (ROFA-insol.) fractions. Sprague-Dawley rats were intratracheally instilled with saline or ROFA-total or ROFA-sol. or ROFA-insol. (1 mg/100 g body wt.). Lung tissue and bronchoalveolar lavage cells were harvested at 4, 24, and 72 h after instillation. Chemiluminescence (CL) of recovered cells was measured as an index of ROS production, and tissue-lipid-peroxidation was assessed to determine oxidative injury. Significant amounts of Al, Fe, and Ni were present in ROFA-sol., whereas ROFA-insol. contained Fe, V, and Al. Using electron spin resonance (ESR), significantly more hydroxyl radicals were measured in ROFA-sol. as compared to ROFA-insol. None of the ROFA samples had an effect on CL or lipid peroxidation at 4 h. Treatment with ROFA-total and ROFA-insol. caused significant increases in both CL (at 24 h) and lipid peroxidation (at 24 and 72 h) when compared to saline control value. ROFA-sol. significantly reduced CL production at 72 h after treatment and had no effect on lipid peroxidation at any time point. In summary, ROFA, particularly its soluble fraction, generated a metal-dependent hydroxyl radical as measured by a cell-free ESR assay. However, cellular oxidant production and tissue injury were observed mostly with the ROFA-total and ROFA-insol. particulate forms. ROS generated by ROFA-sol. as measured by ESR appear not to play a major role in the lung injury caused after ROFA exposure.
pp 19-21 February 2003
In order to identify and quantify free radicals in the tissues of patients with normal physiological and pathological states of births, we developed a method to evaluate the amount of free radicals in myometrium of subplacental area and from body of uterus, using electron spin resonance spectroscopy. Analysis of the concentration of free radicals in the myometrium in full-term pregnancy with normal labour and during uterine inertia was studied. The activities of Ca2+-ATPase, cytochrome c oxidase and succinate dehydrogenase in samples of these tissues were tested too. Low free radical concentrations in these tissues were associated with disturbances in contractile activity of myometrium along with reduction of Ca2+-ATPase, cytochrome c oxidase and succinate dehydrogenase activity. There proved to be an association between the level of free radicals in the tissues and alteration in the physiological processes.
pp 23-27 February 2003
We investigated whether long-term a-tocopherol therapy in chronic smoking affects superoxide generating capacity of neutrophilsex vivo. To this purpose, we randomly assigned 128 male chronic smokers (37 ± 21 pack years of smoking) to treatment with placebo(n = 64) or α-tocopherol (400 IU dL-α-tocopherol daily,n = 64). After two years of therapy, we measured phorbol 12-myristate 13-acetate-induced superoxide production of isolated neutrophils and of diluted whole blood by monitoring reduction of ferricytochrome c and luminol-enhanced peroxidase-catalyzed chemiluminescence. Plasma lipids and lipoproteins were not different between the two treatment groups. As expected, concentrations of a-tocopherol in plasma and in low-density lipoproteins were markedly elevated in the supplemented group compared to the placebo group (+ 120%,P 00001 and + 83%,P 00001, respectively). Consequently, resistance toin vitro oxidation of low-density lipoproteins (reflected by lag time of conjugated diene formation) was higher in the supplemented group than in the placebo group (+ 22%,P 00001). Superoxide generating capacity of neutrophils and superoxide production in diluted whole blood did not differ between α-tocopherol and placebo group. It is concluded that in chronic smoking long-term supranormal α-tocopherol intake does not reduce neutrophil superoxide-anion generating capacity, despite large increases in the concentrations of a-tocopherol in plasma and in low-density lipoproteins.
pp 29-37 February 2003
Exposure of the lung to lipopolysaccharide (LPS) or silica results in an activation of alveolar macrophages μMs), recruitment of polymorphonuclear leukocytes (PMNs) into bronchoalveolar spaces, and the production of free radicals. Nitric oxide (NO) is one of the free radicals generated by bronchoalveolar lavage (BAL) cell populations following either LPS or silica exposure. The purpose of the present study was to assess the relative contributions of AMs and PMNs to the amounts of NO produced by BAL cells following intratracheal (IT) instillation of either LPS or silica. Male Sprague Dawley rats (265-340 g body wt.) were given LPS (10 μg/100 g body wt.) or silica (5 mg/100 g body wt.). BAL cells were harvested 18–24 h post-IT and enriched for AMs or PMNs using density gradient centrifugation. Media levels of nitrate and nitrite (NOx; the stable decomposition products of NO) were then measured 18 h afterex vivo culture of these cells. Following IT exposure to either LPS or silica, BAL cell populations were ∼ 20% AMs and ∼ 80% PMNs. After density gradient centrifugation of BAL cells from LPS or silica-treated rats, cell fractions were obtained which were relatively enriched for AMs (∼ 60%) or PMNs (∼ 90%). The amounts of NOx produced by the AM-enriched fractions from LPS or silica-treated rats were ∼ 2-4-fold greater than that produced by the PMN-enriched fractions. Estimations of the relative contribution of AMs or PMNs to the NOx produced indicated that: (i) following LPS treatment, 75%-89% of the NOx was derived from AMs and 11%-25% from PMNs; and (ii) following silica treatment, 76%-100% of the NOx was derived from AMs and 0.24% from PMNs. Immunohistochemistry for inducible NO synthase on lung tissue sections supported these findings. We conclude that AMs are the major source of the NO produced by BAL cells during acute pulmonary inflammatory responses to LPS or silica.
pp 39-49 February 2003
Insulin resistance is characterized by impaired glucose utilization in the peripheral tissues, accelerated muscle protein degradation, impaired antioxidant defences and extensive cell death. Apparently, both insulin and IGF-1 at physiological concentrations support cell survival by phosphatidylinositol 3 kinase-dependent and independent mechanisms. Postprandial hyperglycemia and hyperinsulinemia are found in insulin resistance, which accompanies the so-called noninsulin dependent diabetes mellitus (diabetes type 2). Evidence also indicates that increased susceptibility of muscle cells and cardiomycoytes to oxidative stress is among the harmful complications of insulin resistance and diabetes. Limited knowledge showing benefits of preconditioning with antioxidants (vitamin C, E, α-lipoic acid, N-acetylcysteine) in order to protect insulin action under oxidative stress prompted the author to discuss the theoretical background to this approach. It should be stressed that antioxidant preconditioning is relevant to prevention of both diabetes- and insulin resistance-associated side-effects such as low viability and cell deletion. Furthermore, antioxidant conditioning promises to provide higher efficacy for clinical applications in myoblast transfer therapy and cardiomyoplasty.
pp 51-55 February 2003
Fly ash was used as a model for ambient particulate matter which is under suspicion to cause adverse pulmonary health effects. The fly ash was pre-sized and contained only particles < 20 μm including an ultrafine fraction (< 100 nm) that contributed 31% to the particle number. In our study, we investigated the influence of fly ash on the promotion of early inflammatory reactions like the formation of reactive oxygen species (ROS) in rat lung epithelial cells (RLE-6TN). Furthermore, we determined the formation of nitric oxide (NO). The cells show a clear dose-response relationship concerning the formation of ROS with regard to the mass of particles applied. Lipopolysaccharide (LPS) added as a co-stimulus did not increase the formation of ROS induced by fly ash. Furthermore, in LPS (01 μg/ml) and tumour necrosis factor-alpha (TNF-alpha; 1 ng/ml) pre-treated cells no increase in reactive oxygen species comparable to fly ash alone is observable. In presence of the metal chelator, desferrioxamine (DFO), ROS formation can be significantly reduced. Neither fly ash nor LPS induced a significant NO release in RLE-6TN cells.
pp 57-60 February 2003
The genotoxicity of reactive oxygen species (ROS) is well established. The underlying mechanism involves oxidation of DNA by ROS. However, we have recently shown that hydrogen peroxide (H2O2), the major mediator of oxidative stress, can also cause genomic damage indirectly. Thus, H2O2 at pathologically relevant concentrations rapidly induces higher order chromatin degradation (HOCD), i.e. enzymatic excision of chromatin loops and their oligomers at matrix-attachment regions. The activation of endonuclease that catalyzes HOCD is a signalling event triggered specifically by H2O2. The activation is not mediated by an influx of calcium ions, but resting concentrations of intracellular calcium ions are required for the maintenance of the endonuclease in an active form. Although H2O2-induced HOCD can efficiently dismantle the genome leading to cell death, under sublethal oxidative stress conditions H2O2-induced HOCD may be the major source of somatic mutations.
pp 61-69 February 2003
This study describes the quantitative relationships between early pulmonary responses and the estimated lungburden or cumulative exposure of respirable-quartz or coal mine dust. Data from a previous bronchoalveolar lavage (BAL) study in coal miners (n = 20) and nonminers (n = 16) were used including cell counts of alveolar macrophages AMs) and polymorphonuclear leukocytes (PMNs), and the antioxidant superoxide dismutase (SOD) levels. Miners’ individual working lifetime particulate exposures were estimated from work histories and mine air sampling data, and quartz lung-burdens were estimated using a lung dosimetry model. Results show that quartz, as either cumulative exposure or estimated lung-burden, was a highly statistically significant predictor of PMN response (P< 00001); however cumulative coal dust exposure did not significantly add to the prediction of PMNs (P = 0.2) above that predicted by cumulative quartz exposure (P < 0.0001). Despite the small study size, radiographic category was also significantly related to increasing levels of both PMNs and quartz lung burden (P-values < 0.04). SOD in BAL fluid rose linearly with quartz lung burden (P < 0.01), but AM count in BAL fluid did not (P > 0.4). This study demonstrates dose-response relationships between respirable crystalline silica in coal mine dust and pulmonary inflammation, antioxidant production, and radiographic small opacities.
pp 71-76 February 2003
Diseases activate the innate immune response which causes ancillary damage to the human body. Peroxynitrite (OONO-) or its carbon dioxide derivatives cause oxidation/nitration and hence mutation to various body polymers e.g. DNA, RNA, protein, lipids and sugars. The control of the ancillary damage can come from antioxidants which inhibit control the amount of peroxynitrite available for damage. In this paper we have developed three different levels of antioxidant screening: (i) Peroxynitrite or SIN-1 reaction with luminol to produce light, and the inhibition of light by substances therefore represents antioxidation. (ii) Nicking of plasmid DNA occurs via oxidants: and is prevented by antioxidants. (iii) Detection of plasmid luciferase activity post-oxidation and infection indicates either prevention or repair of damage: via antioxidants. We found green tea and a number of its polyphenolic constituents effective only at the first level of antioxidation, while extracts of various fruit help at all levels antioxidation. In the final analysis, a combination of green tea extracts and fruits is suggested to produce more complete antioxidant protection.
pp 77-81 February 2003
The present study was carried out to observe the cytotoxicity of yellow sand in comparison with silica and titanium dioxide in a rat alveolar type II cell line (RLE-6TN). Yellow sand (China Loess) was obtained from the loess layer in the Gunsu Province of China. The mean particle diameter of yellow sand was about 0003 ± 0.001 mm. Major elements of yellow sand were Si(27.7 ± 0.6%), Al(6.01± 0.17%), and Ca(5.83 ± 0.23%) in that order. Silica and yellow sand significantly decreased cell viability and increased [Ca2+]i. All three particles increased the generation of H2O2. TiO2 did not change Fenton activity, while silica induced a slight increase of Fenton activity. In contrast, yellow sand induced a significant increase of Fenton activity. Silica, yellow sand and TiO2 induced significant nitrite formations in RLE-6TN cells. Silica showed the highest increase in nitrite formation, while yellow sand induced the least formation of nitrite. Silica and yellow sand increased the release of TNF-α. Based on these results, we suggest that yellow sand can induce cytotoxicity in RLE-6TN cells and reactive oxygen species, Fenton activity and reactive nitrogen species might be involved in this toxicity.
pp 83-89 February 2003
A number of reports indicate the potential for redox signalling via extracellular signal-regulated protein kinases (ERK) during neuronal injury. We have previously found that sustained ERK activation contributes to toxicity elicited by 6-hydroxydopamine (6-OHDA) in the B65 neuronal cell line. To determine whether reactive oxygen species (ROS) play a role in mediating ERK activation and 6-OHDA toxicity, we examined the effects of catalase, superoxide dismutase (SOD1), and metalloporphyrin antioxidants (‘SOD mimetics’) on 6-OHDA-treated cells. We found that catalase and metalloporphyrin antioxidants not only conferred protection against 6-OHDA but also inhibited development of sustained ERK phosphorylation in both differentiated and undifferentiated B65 cells. However, exogenously added SOD1 and heat-inactivated catalase had no effect on either toxicity or sustained ERK phosphorylation. This correlation between antioxidant protection and inhibition of 6-OHDA-induced sustained ERK phosphorylation suggests that redox regulation of ERK signalling cascades may contribute to neuronal toxicity.
pp 91-94 February 2003
Polymorphonuclear leukocytes (PMN) from healthy donors were tested for stimulated release of superoxide anions after being incubated with serum of welders and of a group of unexposed individuals. These two groups were further subdivided either according to age or to smoking habits. The experiments showed that stimulated superoxide production from PMN was inhibited(P 005) by serum from young smokers as compared to that of young nonsmokers, both from the unexposed group. Incubation of PMN with serum from elderly nonsmoking individuals decreased superoxide production as compared to incubation with serum from young nonsmoking individuals, both from the unexposed group. A decrease in superoxide production by incubation with serum of welders as compared to that of unexposed individuals was significant only when the comparison was carried out between the young, non-smoking subgroups. These findings suggest that age, smoking, and exposure to oxidants induce appearance in serum of factors that affect the PMN function.
pp 95-100 February 2003
Coal mining causes health problems, such as pneumoconiosis. We have previously shown that prevalence of pneumoconiosis in workers from various coalmine regions positively correlates with levels of bioavailable iron (BAI) in the coals from that region. In the present study, the nature of reactive oxygen species formed by BAI in the coals and its mechanisms of the induction of biological responses were investigated. Human lung epithelial cell line, A549 cells, were used to examine the induction of interleukin-6 (IL-6), a pro-inflammatory cytokine, which is known to play a crucial role in the development of pneumoconiosis. We found that levels of IL-6 protein as well as its mRNA were significantly increased in the cells treated for 24 h with 20 μg/cm2 of the BAI-containing Pennsylvania (PA) coal; for example we observed 6.7-fold increase in IL-6 protein. Levels of IL-6 protein in cells treated with the Utah (UT) coal containing low-BAI were only 1.9-fold of the control levels. The enhancing effect on the IL-6 by the PA coal was similar to that caused by hydrogen peroxide. Superoxide dismutase (SOD), catalase (CAT), and N-acetyl-L-cysteine (NAC) all had inhibitory effects on the PA coal-induced IL-6 formation. However, CAT had the least protective effect as compared to SOD and NAC. Our results indicate that BAI in the PA coal may induce IL-6 through both ferryl species (via iron autoxidation) and hydroxyl radicals (via the Fenton/Haber Weiss reactions).
pp 101-107 February 2003
This investigation focuses on the application of anin vitro assay in elucidating the role of lung lining fluid antioxidants in the protection against inhaled particles, and to compare the toxicities of different airborne particulate matter (PM), PM10, collections from South Wales, UK.
PM collections from both urban and industrial sites caused 50% oxidative degradation of DNAin vitro at concentrations as low as 12.9 ± 2.1 μ ml-1 and 4.9 ± 0.9 μg ml-1 respectively. The primary source of this bioreactivity was found to be the soluble fraction of both particle collections. The coarser PM10_2.5 fraction also showed greater oxidative bioreactivity than the PM2.5_0.1 in both cases. When repeated in the presence of a low molecular weight fraction of fresh pulmonary lavage fluid, as well as in artificial lung lining fluid (200 (μM urate, glutathione and ascorbate), the DNA damage was significantly reduced in all cases(P 0.05). The antioxidants exerted a greater effect on the industrial samples than on the urban samples, and on the PM10_2.5 fractions than on the PM2.5_0.1 fractions, supporting the previous findings that respirable PM and urban samples contain fewer free radical sources than inhalable PM and industrial samples.
pp 109-113 February 2003
Analysis of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor produced by the cytochrome P450 pathway, presently requires high-performance liquid chromatography (HPLC) and gas chromatography/ mass spectrometry (GC/MS). To simplify 20-HETE analysis, competitive ELIS As were developed using polyclonal anti-20-HETE coated ELISA plates to which free 20-HETE and 20-HETE conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) were added. Assays were developed with and without a proprietary enhancer solution which allows for the extraction-free measurement of 20-HETE in urine samples. The bound 20-HETE-HRP or 20-HETE-AP was detected using 3,3′,5,5,-tetramethylbenzidine and p-nitrophenyl phosphate, respectively. Sensitivities expressed as 80% B/B0, were 01 ng/ml for the HRP assay, and 0–5 ng/ml for the AP assay, withr2 = 0.99 for both formats. Of the 17 lipids tested for cross-reactivity, arachidonic acid showed the highest (0.32%) followed by racemic 5-HETE (007%) and 8,9-dihydroxyeicosatrienoic acid (DHET) (004%). Preliminary validation experiments examining serum and urine concentrations of 20-HETE yield values that fall within the ranges established by GC/MS in the literature. These ELISAs provide simple and inexpensive methods for the analysis of 20-HETE in biological samples.
pp 115-119 February 2003
A simple, fast, precise and biologically relevant toxicity assay for screening cytotoxicity of minerals would have distinct advantages due to its cost benefits and relative savings in time. Furthermore, a bioassay to differentiate acute and chronicin vivo pulmonary reactions could have potential value as predictors of fibrogenicity and pathogenicity. In this study we examined the potential use of lucigenin as a probe to evaluate the correlation between chemiluminescence (CL) generated by alveolar macrophages with the known cytotoxicity and pathogenicity by conventional bioassays. In this study, we used small doses of dust (20 μg) to minimize cellular overload and to maintain homeostasis. Crystalline silica a highly fibrogenic dust was used as positive control and results are compared with those for bentonite, kaolin and talc. Among the three minerals compared with silica, bentonite was more reactive (27%) in CL assay and declined sharply compared to other minerals. This sudden decline in bentonite CL is caused by cytotoxicity leading to cell death. CL-induced by talc was comparable to silica and declines slowly. Kaolin on the other hand produced relatively a weaker (25%) CL compared to silica. Our data using relatively low doses of dust suggest that the CL assay may have a better predictive value in cytotoxicity evaluations compared to conventional toxicity assays.
pp 121-128 February 2003
Increasing evidence supports the role of excitotoxicity in neuronal cell injury. Thus, it is extremely important to explore methods to retard or reverse excitotoxic neuronal injury. In this regard, certain dietary compounds are begining to receive increased attention, in particular those involving phytochemicals found in medicinal plants in alleviating neuronal injury. In the present study, we examined whether medicinal plant extracts protect neurons against excitotoxic lesions induced by kainic acid (KA) in female Swiss albino mice. Mice were anesthetized with ketamine and xylazine (200 mg and 2 mg/kg body wt. respectively) and KA (0.25 μg in a volume of 0.5 μ1) was administered to mice by intra hippocampal injections. The results showed an impairment of the hippocampus region of brain after KA injection. The lipid peroxidation and protein carbonyl content were significantly(P 005) increased in comparison to controls. Glutathione peroxidase (GPx) activity (EC 1.11-1.9) and reduced glutathione (GSH) content declined after appearance of excitotoxic lesions. As GPx and GSH represent a major pathway in the cell for metabolizing hydrogen peroxide (H2O2), their depletion would be expected to allow H2O2 to accumulate to toxic levels. Dried ethanolic plant extracts ofWithania somnifera (WS),Convolvulus pleuricauas (CP) andAloe vera (AV) dissolved in distilled water were tested for their total antioxidant activity. The diet was prepared in terms of total antioxidant activity of plant extracts. The iron (Fe3+) reducing activity of plant extracts was also tested and it was found that WS and AV were potent reductants of Fe3+ at pH 5.5. CP had lower Fe3+ reducing activity in comparison to WS and AV. Plant extracts given singly and in combination 3 weeks prior to KA injections resulted in a decrease in neurotoxicity. Measures of lipid peroxidation and protein carbonyl declined. GPx activity and GSH content were elevated in hippocampus supplemented with WS and combination of WS + CP + AV. However, when CP and AV were given alone, the changes in the GPx activity and GSH content were not significant. Although the major factors involved in these properties of phytochemicals remain to be specified, the finding of this study has suggested that phytochemicals present in plant extracts mitigate the effects of excitotoxicity and oxidative damage in hippocampus and this might be accomplished by their antioxidative properties.
pp 129-134 February 2003
Effect of lipopolysaccharide (LPS) on RAW264.7 macrophage cell line was studied. LPS-treated RAW264.7 cells increased in cell size and acquired distinct dendritic morphology. At the optimal dose of LPS (1 μg/ml), almost 70% RAW264.7 cells acquired dendritic morphology. Flow cytometric studies indicate that the cell surface markers known to be expressed on dendritic cells and involved in antigen presentation and T cell activation (B7.1, B7.2, CD40, MHC class II antigens and CD1d) were also markedly upregulated on LPS-treated RAW264.7 cells. Our results suggest the possibility that LPS by itself could constitute a sufficient signal for differentiation of macrophages into DC-like cells.
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