Intracellular Superoxide Anion Levels Research Articles

Epigenetic silencing of GCH1promotes hepatocellular carcinoma growth by activating superoxide anion-mediated ASK1/p38 signaling via inhibiting tetrahydrobiopterin de novo biosynthesis

Metabolic reprogramming is a hallmark of cancer, including hepatocellular carcinoma (HCC). However, its role in HCC remains to be elucidated. Herein, we identified GTP cyclohydrolase 1 (GCH1), the first rate-limiting enzyme in tetrahydrobiopterin (BH4) de novo biosynthesis, as a novel metabolic regulator of HCC. GCH1 was frequently down-regulated in HCC tissues and cell lines by promoter methylation. Low GCH1 expression was associated with larger tumor size, increased tumor number, and worse prognosis in two independent cohorts of HCC patients. Functionally, GCH1 silencing promoted HCC growth in vitro and in vivo, while GCH1 overexpression exerted an opposite effect. The metabolite BH4 inhibited HCC growth in vitro and in vivo. GCH1 silencing exerted its growth-promoting effect through directly inhibiting BH4 de novo biosynthesis. Mechanistically, GCH1 silencing activated ASK1/p38 signaling; pharmacological or genetic inhibition of ASK1 or p38 abolished GCH1 silencing-induced growth-promoting effect. Further mechanistic studies found that GCH1 silencing-induced BH4 reduction resulted in an increase of intracellular superoxide anion levels in a dose-dependent manner, which mediated the activation of ASK1/p38 signaling. Collectively, our study reveals that epigenetic silencing of GCH1 promotes HCC growth by activating superoxide anion-mediated ASK1/p38 signaling via inhibiting BH4 de novo biosynthesis, suggesting that targeting GCH1/BH4 pathway may be a promising therapeutic strategy to combat HCC.

Icariin Prevents Extracellular Matrix Accumulation and Ameliorates Experimental Diabetic Kidney Disease by Inhibiting Oxidative Stress via GPER Mediated p62-Dependent Keap1 Degradation and Nrf2 Activation.

The present study aimed to determine whether icariin could attenuate type 1 diabetic nephropathy (T1DN) induced by streptozotocin (STZ) after 4 weeks or not. Therefore, its therapeutic effect on diabetic kidney disease was investigated in view of reactive oxygen (ROS) and extracellular matrix (ECM) generation in human glomerular mesangial cells under high glucose. To establish the participation and the key role of GPER and Nrf2 in ECM deposition, a combination of G15 (antagonist of GPER) or siGPER and siNrf2 were performed, respectively. The results showed that T1DN can be significantly inhibited by oral icariin, evidenced by improvement of 24 h urinary volume, 24 h proteinuria, microalbuminuria, and histopathological changes of kidney. Icariin decreased the levels of intracellular superoxide anion, impeded the generation of fibronectin and increased the expression and activity of antioxidant enzymes in the human glomerular mesangial cells treated with high glucose. It acted as a GPER activator, increased dissociation of Nrf2/Keap1 complexes, combination of Keap1/p62 complexes, Nrf2 translocation to nuclear, Nrf2/ARE DNA binding activity, and ARE luciferase reporter gene activity in glomerular mesangial cells. The Nrf2 inhibitor ML385 or siNrf2 obviously abolished extracellular matrix (ECM) generation inhibited by icariin. Furthermore, icariin-induced Nrf2 activation was mainly dependent on p62-mediated Keap1 degradation, which functions as an adaptor protein during autophagy. The GPER antagonist G15 and siGPER obviously abolished the above effects by icariin. Taken together, the present study demonstrated that the therapeutic effects of icariin on type 1 diabetic nephropathy in rats via GPER mediated p62-dependent Keap1 degradation and Nrf2 activation.

Effects of red-light irradiation on the function and survival of fresh and liquid-stored donkey semen

This study sought to determine whether sperm irradiation using a light emission diode (LED) at 620–630 nm affects the motility, membrane integrity (viability), mitochondrial activity and intracellular levels of reactive oxygen species (ROS) in fresh diluted and liquid-stored donkey semen. With this purpose, sixteen ejaculates (eight fresh diluted and eight cooled-stored) were collected from eight adult jackasses. Fresh semen samples were diluted in Kenney extender and stimulated with red-light after collection, whereas cooled semen was stored at 4 °C for 24 h after dilution and then irradiated. In all cases, semen samples were packed into 0.5-mL transparent straws, which were then randomly divided into control and 19 treatments: six consisted of single red-light exposure, and the other 13 involved irradiation at light-dark-light intervals. Upon irradiation, sperm motility, membrane integrity mitochondrial membrane potential (MMP) and intracellular levels of superoxide anion (·O2−) and hydrogen peroxide (H2O2) were evaluated. While specific light-patterns increased both sperm motility and mitochondrial activity, they did not affect sperm membrane integrity and had no clear impact on intracellular ROS levels. The effects of irradiation patterns differed between fresh and cooled semen since, whereas 1 and 4 min patterns induced the greatest increments in the total and progressive motility of fresh semen, 4 min, 4-1-4 and 4-4-4 were the most suitable for cooled-stored samples. In both fresh diluted and cooled-stored semen, the motility increase observed after light-stimulation for 4 min was concomitant with changes in the percentages of spermatozoa with high mitochondrial membrane potential. In summary, this study shows, for the first time, that specific irradiation patterns increase sperm motility and mitochondrial activity in the donkey. Furthermore, the precise effect of red-light appears to depend on the specific functional status of cells, with separate effects on fresh and cooled samples.

Action of Cholesterol-Loaded Cyclodextrin on Viability of Ram Semen

Background: Studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some cases can also remove this cholesterol from the plasma membrane. The mechanism of action of CLC is not well understood, however, it seems to involve sperm protection during the freezing and thawing process. Studies show that its use enhancing increased osmotic tolerance and reduced premature sperm capacitation reaction. In this sense, studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some cases can also remove this cholesterol from the plasma membrane. Improvements were reported in the sperm parameters of buffaloes, bulls, stallions and sheep. Ram naturally present less lipids in their membrane, on average 27%, while bulls have 31%, rabbits 62%, and humans 50%. The aimed of the present study was to evaluate the use of cholesterol-loaded cyclodextrin (CLC), a commercial diluent, in the kinetics and viability of frozen and thawed ram spermatozoa. Materials, Methods & Results: Five ejaculates, from five rams of Dorper breed were collected and divided into three groups: control, 1 mg CLC and 2 mg CLC. Semen was diluted in different concentrations of CLC (0, 1, and 2 mg/120×106 spermatozoa), and incubated at room temperature (21°C) for 10 min. Samples were conditioned in 0.5 mL straws and incubated at 5°C for 4 h, exposed to LN2 vapor for 10 min and storing a cryogenic container. The parameters as spermatic kinetics, plasma membrane, acrosomal membrane (MPAI, %), and intracellular levels of superoxide anion (O2-) were evaluated. Sperm progressive motility (PM), rapid spermatozoa percentage (RAP), linearity (LIN, %), average path velocity (VAP, μm/s) and MPAI (%) were more satisfactory with the use of 1 mg compared to 2 mg (P < 0.05). In addition, 1 mg CLC showed decreased levels of superoxide anion formation (O2-), a free radical detrimental to spermatozoa (P < 0.05). The use of 2 mg of CLC reduce VAP (P < 0.05) and did not have any beneficial effect on the evaluated parameters. Discussion: Authors did not observe improvement in the parameters of progressive motility when using 1 mg of CLC in goat semen and 2 mg in bull semen with the slow freezing protocol. This differs from our work, as we found that 1 mg of CLC improved the PM parameters, but not at the concentration of 2 mg CLC. Additionally, authors verified that cyclodextrin at 3 mg concentration was effective in protecting the sperm against the deleterious effects of H2O2. They obtained superior plasma membrane motility, viability, and integrity of the CLC-treated samples compared to the control group. The superoxide anion (O2-) is a free radical formed from molecular oxygen by the addition of an electron. It is generated spontaneously, mainly in the membrane of the mitochondria, by the respiratory chain and by flavoenzimes, lipoxygenases, and cicloxygenases. In our study, we found a difference between the study group with 1 mg CLC and the control group. Thus, we suggest that CLC may have a beneficial effect in stabilizing the sperm plasma membrane. Use of CLC at a concentration of 1 mg was found to be effective for the improvement of parameters of sperm progressive motility, rapid sperm percentage, and plasma and acrosomal membrane integrity. In addition, the study group with 1 mg of CLC showed decreased levels of superoxide anion formation, a free radical detrimental to spermatozoa.

Bergenin impedes the generation of extracellular matrix in glomerular mesangial cells and ameliorates diabetic nephropathy in mice by inhibiting oxidative stress via the mTOR/β-TrcP/Nrf2 pathway

Bergenin, a plant polyphenol, has been reported to lower the blood glucose level and ameliorate kidney function in streptozotocin (STZ)-induced diabetic rats. Herein, its protective effect on diabetic nephropathy (DN) was explored in view of extracellular matrix (ECM) generation in glomerular mesangial cells. Glomerular mesangial cells were treated with high glucose, and Q-PCR as well as western blot were used to determine the expression of ECM. To establish the participation and role of mammalian target of rapamycin (mTOR) and nuclear factor erythroid-derived 2-related factor 2 (Nrf2) in ECM generation, a combination of l-leucine (activator of mTOR) and Nrf2 shRNA transfection were performed, respectively. Moreover, a DN model was established in mice using high-glucose/high-fat diet and STZ. Bergenin impeded the generation of TGF-β1 and ECM, decreased the levels of intracellular superoxide anion and hydrogen peroxide, and increased the activity of antioxidant enzymes in the glomerular mesangial cells (HBZY-1 and HRMC cells) treated with high glucose. The inhibition of ECM generation by bergenin was dependent on the down-regulation of oxidative stress as confirmed via a superoxide overexpression system. The activation of Nrf2 was required for bergenin to inhibit the oxidative stress and ECM generation. Moreover, bergenin was found to inhibit the phosphorylation of mTOR, which is located at the upstream of Nrf2. Bergenin did not interfere with the expression of Nrf2 mRNA and Keap1 (the classic degradation control factor of Nrf2), but markedly inhibited the protein expression of the β-TrcP, an effect which could be abolished by l-leucine. In DN model mice, l-leucine diminished the ability of bergenin to reduce the levels of superoxide anion, hydrogen peroxide and ECM, which contributed to the eradication of the ameliorative effect of bergenin on nephropathy. Bergenin can inhibit glucose-induced ECM production in glomerular mesangial cells through the down-regulation of oxidative stress via the mTOR/β-TrcP/Nrf2 pathway, and it might be a candidate drug for the prevention and treatment of DN.

Redox-modulating agents target NOX2-dependent IKKε oncogenic kinase expression and proliferation in human breast cancer cell lines

Oxidative stress is considered a causative factor in carcinogenesis, but also in the development of resistance to current chemotherapies. The appropriate usage of redox-modulating compounds is limited by the lack of knowledge of their impact on specific molecular pathways. Increased levels of the IKKε kinase, as a result of gene amplification or aberrant expression, are observed in a substantial number of breast carcinomas. IKKε not only plays a key role in cell transformation and invasiveness, but also in the development of resistance to tamoxifen. Here, we studied the effect of in vitro treatment with the redox-modulating triphenylmethane dyes, Gentian Violet and Brilliant Green, and nitroxide Tempol on IKKε expression and cell proliferation in the human breast cancer epithelial cell lines exhibiting amplification of IKKε, MCF-7 and ZR75.1. We show that Gentian Violet, Brilliant Green and Tempol significantly decrease intracellular superoxide anion levels and inhibit IKKε expression and cell viability. Treatment with Gentian Violet and Brilliant Green was associated with a reduced cyclin D1 expression and activation of caspase 3 and/or 7. Tempol decreased cyclin D1 expression in both cell lines, while activation of caspase 7 was only observed in MCF-7 cells. Silencing of the superoxide-generating NOX2 NADPH oxidase expressed in breast cancer cells resulted in the significant reduction of IKKε expression. Taken together, our results suggest that redox-modulating compounds targeting NOX2 could present a particular therapeutic interest in combination therapy against breast carcinomas exhibiting IKKε amplification.

Synergism between arsenite and proteasome inhibitor MG132 over cell death in myeloid leukaemic cells U937 and the induction of low levels of intracellular superoxide anion

Increased oxygen species production has often been cited as a mechanism determining synergism on cell death and growth inhibition effects of arsenic-combined drugs. However the net effect of drug combination may not be easily anticipated solely from available knowledge of drug-induced death mechanisms. We evaluated the combined effect of sodium arsenite with the proteasome inhibitor MG132, and the anti-leukaemic agent CAPE, on growth-inhibition and cell death effect in acute myeloid leukaemic cells U937 and Burkitt's lymphoma-derived Raji cells, by the Chou–Talalay method. In addition we explored the association of cytotoxic effect of drugs with changes in intracellular superoxide anion (O 2 −) levels. Our results showed that combined arsenite + MG132 produced low levels of O 2 − at 6 h and 24 h after exposure and were synergic on cell death induction in U937 cells over the whole dose range, although the combination was antagonistic on growth inhibition effect. Exposure to a constant non-cytotoxic dose of 80 μM hydrogen peroxide together with arsenite + MG132 changed synergism on cell death to antagonism at all effect levels while increasing O 2 − levels. Arsenite + hydrogen peroxide also resulted in antagonism with increased O 2 − levels in U937 cells. In Raji cells, arsenite + MG132 also produced low levels of O 2 − at 6 h and 24 h but resulted in antagonism on cell death and growth inhibition. By contrast, the combination arsenite + CAPE showed high levels of O 2 − production at 6 h and 24 h post exposure but resulted in antagonism over cell death and growth inhibition effects in U937 and Raji cells. We conclude that synergism between arsenite and MG132 in U937 cells is negatively associated to O 2 − levels at early time points after exposure.

Improvement of leucocyte functions in mature and old mice after 15 and 30 weeks of diet supplementation with polyphenol-rich biscuits

To study the effect of diet supplementation with polyphenols on several functions suffering age-related changes, in peritoneal leucocytes from mature and old mice. Five groups of female ICR mice were used. Four groups received a supplementation (20% wt/wt) of biscuits with different cereal fractions naturally rich in polyphenols (named CO49, CO50, CO52, CO53), containing different amounts of catechin, p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, sinapic acid, ferulic acid, rutin and oryzanol. The control group received only standard maintenance diet. Peritoneal suspensions were obtained after 15 and 30 weeks of diet supplementation, when the age of the animals was 49 ± 2 (mature mice) and 64 ± 2 weeks (old mice), respectively. The functions analysed were: chemotaxis of macrophages and lymphocytes, phagocytosis of particles by macrophages, intracellular superoxide anion levels, lymphoproliferative response to mitogens (concanavalin A and lipopolysaccharide), interleukin-2 secretion and natural killer (NK) activity, as functions that decrease with age, and adherence of macrophages and lymphocytes and tumour necrosis factor-α secretion as functions with age-related increase. The supplementation, in general, increased the functions that decrease with age and decreased those that increase with age. There were differences in the effects shown by the four kinds of biscuits depending on the function studied and the number of weeks of supplementation. Since the immune system has been proposed as a good marker of health and predictor of longevity, diet supplementation with cereals naturally rich in polyphenols could be an important way for health preservation with age and reaching high longevity.

Crucial role of cytochrome P450 in hepatotoxicity induced by 2,3‐dimethoxy‐1,4‐naphthoquinone in rats

Quinone toxicity is induced by two principal mechanisms: arylation/alkylation and a redox cycle. We have previously shown that increases in intracellular levels of superoxide anion and cell death induced by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a redox cycling quinone, are enhanced by pretreatment of rat primary hepatocytes with cytochrome P450 inhibitors. This indicates a novel interaction of quinones with cytochrome P450, and is thus worthy of further investigation using an in vivo model. The aim of this study was to examine the effects of cytochrome P450 inhibitors on DMNQ-induced hepatotoxicity in rats. When DMNQ was administered intraperitoneally, the activities of serum alanine aminotransferase and aspartate aminotransferase were found to increase in a dose-dependent manner, indicating that hepatotoxicity was induced by treatment with DMNQ. Pretreatment with the cytochrome P450 inhibitors SKF-525A (SKF), cimetidine and ketoconazole potentiated the DMNQ-induced hepatotoxicity. The blood concentration of DMNQ was not affected by administration of SKF. Pretreatment with the antioxidant α-tocopherol almost completely attenuated the hepatotoxicity induced by DMNQ and by the combination of DMNQ with SKF. Levels of reduced glutathione in the liver were decreased and levels of oxidized glutathione were increased by treatment with DMNQ. These effects were potentiated by pretreatment with SKF. DMNQ-induced lipid peroxidation in the liver was also enhanced by pretreatment with SKF. Taken together, these results indicate that DMNQ-induced hepatotoxicity is augmented by inhibition of cytochrome P450 and that this augmentation is due to the enhancement of oxidative stress.

Neutrophils of Centenarians Show Function Levels Similar to Those of Young Adults

To analyze several functions and antioxidant parameters of peripheral blood neutrophils from healthy centenarians (men and women) and compare them with those of healthy young (aged 25-35) and middle-aged (aged 65-75) men and women. Cross-sectional study. Community-based. Twenty-one healthy centenarians (8 men), 30 young adults (15 men), and 30 middle-aged adults (15 men). Several neutrophil functions (adherence, chemotaxis, phagocytosis, and stimulated and nonstimulated intracellular superoxide anion levels) and antioxidant parameters (glutathione levels and catalase activity) were measured in peripheral blood neutrophil suspension in the three study groups. Neutrophil functions of the middle-aged group were worse than those of young adults and centenarians (lower chemotaxis and phagocytosis and higher adherence and superoxide anion levels). The neutrophil functions of the centenarians were closer to those of the young adults. Age-related differences in neutrophil functions were fundamentally similar in men and women, except for intracellular superoxide anion levels, which were lower in young adult women than in young adult men. With normal aging, total glutathione levels decrease, but the centenarians in this study showed levels similar to those of young adults. Centenarians showed the highest catalase activity of the three groups. Progressive impairment of the immune system accompanies aging. The better preservation of function and antioxidant systems in the neutrophils of centenarians could play a key role in the longevity of these subjects.

Chromium (VI) induces insulin resistance in 3T3-L1 adipocytes through elevated reactive oxygen species generation

Reactive oxygen species (ROS) have been proposed to be involved in the development of insulin resistance, although the exact molecular link between ROS and insulin resistance remains to be determined. Chromium (Cr(VI)) is known as an inducer of ROS. Therefore, this study examined whether Cr(VI) could induce insulin resistance. It demonstrated that Cr(VI) treatment significantly inhibited insulin-stimulated glucose uptake and attenuated insulin signalling. Moreover, Cr(VI) treatment markedly increased the intracellular levels of superoxide anion, hydrogen peroxide and hydroxyl radical. N-acetylcysteine, superoxide dismutase and catalase can block the ROS generation and alleviate the insulin resistance induced by Cr(VI) treatment. In addition, Cr(VI) treatment induced endoplasmic reticulum (ER) stress and JNK activation and these effects were diminished by N-acetylcysteine. These results suggested that ROS generation through Cr(VI) treatment cause ER stress, JNK activation and insulin resistance in adipocytes. Therefore, the oxidative stress could be a potential interventional target for insulin-resistance related diseases.

Folic acid inhibits homocysteine-induced superoxide anion production and nuclear factor kappa B activation in macrophagesThis paper is one of a selection of papers published in this Special Issue, entitled Young Investigator's Forum.

Folic acid supplementation is a promising approach for patients with cardiovascular diseases associated with hyperhomocysteinemia. We have demonstrated that homocysteine (Hcy) activates nuclear factor-kappaB (NF-kappaB), a transcription factor that plays an important role in inflammatory responses. The aim of the present study was to investigate the effect of folic acid on Hcy-induced NF-kappaB activation in macrophages. Hcy treatment (100 micromol/L) resulted in NF-kappaB activation and increased monocyte chemoattractant protein-1 (MCP-1) expression in THP-1 derived macrophages. Hcy-induced NF-kappaB activation was associated with a significant increase in the intracellular superoxide anion levels. There was a significant increase in phosphorylation and membrane translocation of NADPH oxidase p47phox subunit in Hcy-treated cells. Addition of folic acid (200 ng/mL) to the culture medium abolished NADPH oxidase-dependent superoxide anion generation in macrophages by preventing phosphorylation of p47phox subunit. Consequently, Hcy-induced NF-kappaB activation and MCP-1 expression was inhibited. Such an inhibitory effect of folic acid was independent of its Hcy-lowering ability. Taken together, these results suggest that folic acid treatment can effectively inhibit Hcy-induced oxidative stress and inflammatory responses in macrophages. This may represent one of the mechanisms by which folic acid supplementation exerts a protective effect in cardiovascular disorders.

In vitro response of the striped bass natural resistance-associated macrophage protein, Nramp, to LPS and Mycobacterium marinum exposure

Mycobacteriosis in Chesapeake Bay (USA) striped bass Morone saxatilis is an ongoing disease problem with important economic implications for a large commercial and recreational fishery. Additionally, striped bass serve as a reservoir of potential mycobacterial zoonoses. Recently, we described a striped bass gene homolog of the natural resistance-associated macrophage protein family (MsNramp), which is responsible for resistance to mycobacterial infections in mice. Striped bass MsNramp is strongly induced in peritoneal exudate cells (PE) in vivo after intraperitoneal injection with Mycobacterium spp. The purpose of the present study was to investigate short-term in vitro MsNramp expression and reactive oxygen intermediate (ROI) production in primary cultures of adherent PE after exposure to bacterial lipopolysaccharide (LPS), or live- or heat-killed (HK) Mycobacterium marinum. PE expressed significantly higher levels of MsNramp at 4 and 24 h post-treatment with live and HK M. marinum. MsNramp response to LPS was dose-dependent in these cells, with maximum expression at 4 h and 20 μg/ml LPS. Treatment of PE with LPS resulted in increased intracellular superoxide anion levels, whereas treatment with live M. marinum caused a significant depression. This study is the first report of induction of a teleost Nramp in vitro by mycobacteria, and supports findings of teleost Nramp induction by LPS.

Effect of two phenanthrene alkaloids on angiotensin II-induced leukocyte-endothelial cell interactions in vivo.

1. The present study has evaluated the effect of two phenanthrene alkaloids, uvariopsine and stephenanthrine, on angiotensin II (Ang-II)-induced leukocyte-endothelial cell interactions in vivo and the mechanisms involved in their activity. Intravital microscopy within the rat mesenteric microcirculation was used. 2. A 60 min superfusion with 1 nm Ang-II induced a significant increase in the leukocyte-endothelial cell interactions that were completely inhibited by 1 microm uvariopsine cosuperfusion. A lower dose of 0.1 microm significantly reduced Ang-II-induced leukocyte adhesion by 75%. 3. When Ang-II was cosuperfused with 1 and 0.1 microm stephenanthrine, Ang-II-induced leukocyte responses were significantly diminished. A lower dose of 0.01 microm only affected Ang-II-induced leukocyte adhesion. 4. Both alkaloids inhibited Ang-II-induced endothelial P-selectin upregulation and the generation of reactive oxygen species (ROS) in endothelial cells stimulated with Ang-II, in fMLP-stimulated human neutrophils (PMNs) and in the hypoxanthine-xanthine oxidase system. However, cyclic AMP levels in PMNs stimulated with fMLP were not affected. 5. Uvariopsine and stephenanthrine inhibited PAF-induced elevations in intracellular calcium levels in PMNs (IC50 values: 15.1 and 6.1 microm respectively) and blocked the binding of [3H]PAF to these leukocytes. They also reduced PAF-induced increases in intracellular levels of superoxide anion and hydrogen peroxide. 6. In conclusion, stephenanthrine and uvariopsine are potent inhibitors of Ang-II-induced leukocyte accumulation in vivo. This effect appears to be mediated through ROS scavenging activity and blockade of PAF receptor. Thus, they have potential therapeutic interest for the control of leukocyte recruitment that occurs in cardiovascular disease states in which Ang-II is involved.

Aberrant redox regulation in human metastatic melanoma cells compared to normal melanocytes

Melanocytes and melanoma cells contain melanin, a complex polymer that modulates redox changes in these cells. Relative intracellular hydrogen peroxide levels measured by dichlorodihydrofluorescein are similar in the two cell types, but the levels of superoxide anion measured by dihydroethidium were markedly increased in melanoma cells. Chelator-induced oxidative stress is efficiently suppressed by melanocytes without substantial recruitment of the transcription factors NF-κB and AP-1 as measured by electrophoretic mobility shift assay and quantitated by densitometry or by a change in frequency of apoptosis as determined by annexin V binding. In contrast, NF-κB in melanoma cells is strongly recruited by changes in redox status and exhibits a correlative relationship to intracellular hydrogen peroxide (but not superoxide anion). However, the response of the NF-κB pathway to intracellular hydrogen peroxide is anomalous, including downregulation of p65 and IκBα RNA expression (Northern blot). Additionally, recruitment of AP-1 binding in melanoma cells was directly correlated with intracellular levels of superoxide anion (but not hydrogen peroxide). Neither the degree of NF-κB nor AP-1 binding in melanoma cells was related to the frequency of apoptosis. The responsiveness of NF-κB and AP-1 recruitment to intracellular levels of hydrogen peroxide and superoxide anion without concomitant control of apoptosis provides a general mechanism by which these cells can escape noxious injury (e.g., chemotherapy). The marked enhancement of apoptosis in melanoma cells by chelators indicates, however, that this alteration can be circumvented and offers a unique therapeutic window to explore.

Cadmium-induced cell transformation and tumorigenesis are associated with transcriptional activation of c-fos, c-jun, and c-myc proto-oncogenes: role of cellular calcium and reactive oxygen species.

The molecular mechanisms of carcinogenesis by cadmium were studied using BALB/c-3T3 cell transformation and nude mouse tumorigenesis models. BALB/c-3T3 cells transformed with cadmium chloride were subcutaneously injected into nude mice to develop tumors and the cell lines derived from these tumors were used in the present study. The proto-oncogenes c-fos and c-jun were overexpressed in 100% (10 out of 10) of the cell lines, while a statistically significant overexpression of c-myc was observed in 40% (4 out of 10) of the cell lines. Analysis of tumor cells stained with fluorescent dyes specific for reactive oxygen species revealed that these cells possessed markedly higher levels of superoxide anion and hydrogen peroxide compared with the nontransformed cells. Similarly, the intracellular calcium level was higher in the tumor cells compared with the nontransformed cells. Overexpression of the proto-oncogenes in these cells was blocked by treating the cells with superoxide dismutase, catalase, and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra acetoxy methyl ester (BAPTA/AM), which are scavengers of superoxide anion, hydrogen peroxide, and calcium, respectively. This confirmed that the overexpression of the proto-oncogenes in the tumor cells required elevated intracellular levels of reactive oxygen species and calcium. In addition to the scavengers of reactive oxygen species and calcium, inhibitors specific for transcription (actinomycin D), protein kinase C (RO-31-8220), and MAP kinase (PD 98059) also blocked the cadmium-induced overexpression of the proto-oncogenes in the tumor cells. Exposure of the nontransformed BALB/c-3T3 cells to 20 microM cadmium chloride for 1 h caused elevated intracellular levels of superoxide anion, hydrogen peroxide, and calcium, with corresponding increases in the expression levels of c-fos, c-jun, and c-myc. As in the case of the tumor cells, treating the nontransformed cells with the various modulators prior to their exposure to cadmium chloride resulted in inhibition in the expression of the proto-oncogenes. Based on these data, we conclude that the cadmium-induced overexpression of cellular proto-oncogenes is mediated by the elevation of intracellular levels of superoxide anion, hydrogen peroxide, and calcium. Further, the cadmium-induced overexpression of the proto-oncogenes is dependent on transcriptional activation as well as on pathways involving protein kinase C and MAP kinase.

Changes with aging in the modulation of macrophages by norepinephrine

The effect of aging on the norepinephrine (NE)-induced modulation of phagocytic and oxygen-dependent microbicidal processes of mouse peritoneal macrophages was studied. Phagocytosis of latex beads on culture plates and superoxide anion production was evaluated in young (12 weeks), adult (22 weeks), mature (48 weeks) and old (72 weeks) BALB/c mice after in vitro incubation with 10 −12, 10 −9, 10 −7, 10 −5 or 10 −3 M concentrations of NE. The results indicate that the phagocytic response to NE is quite similar in young and mature mice, with increased phagocytosis after incubation with 10 −7 and 10 −3 M, and decreased phagocytosis with 10 −5 M. Macrophages from adult mice increased their phagocytic capacity after incubation with the highest concentrations of NE (10 −5 and 10 −3 M) and macrophages from old animals only were stimulated with 10 −3 M. In addition, it was found that usually NE increased the extracellular superoxide anion production in the absence of phagocytosis in adult mice. No statistically significant changes were found in intracellular superoxide anion levels, but an increase was seen after phagocytosis in macrophages from adult, mature and old animals, especially after incubation with 10 −5 M. In conclusion, the results obtained indicate that the modulation of macrophages by NE does not only depend on the concentration of this neurotransmitter, but also on age.