Long-term UVA eye irradiation causes decreased learning ability in mice.

BACKGROUND: Long-term eye radiation with ultraviolet A (UVA) denatures the cells of the cerebral hippocampus. However, the influence on memory and learning ability in mice is not known. METHODS: HR-1 mice were used. We irradiated the eyes or dorsal skin of the mice with UVA at a dose of 110 kJ/m(2) using an FL20SBLB-A lamp for 6, 12, and 24 months. RESULTS: The mean escape latency in the water maze was significantly increased in the UVA-irradiated mice in comparison with that seen in the controls. In the mice in which UVA eye irradiation was performed for 24 months, the depression of memory and learning ability was remarkable. The acetylcholinesterase activity, choline acetyltransferase activity, and acetylcholine content in the brain in the UVA eye-irradiated mice were significantly less than those observed in the control mice. Furthermore, during UVA eye irradiation, the levels of ß-amyloid (Aß), γ-secretase, which produces Aß peptide, and advanced glycation end products increased. Moreover, the effects of UVA eye irradiation increased with the duration of irradiation (or aging), and the introduction of glucose into the brain also decreased with UVA eye irradiation. CONCLUSIONS: These results indicate that UVA eye irradiation induces a decreased memory and learning ability in mice.

Role of adrenocorticotropic hormone in the modulation of pollinosis induced by pollen antigens.

BACKGROUND: A mild restraint stressor suppressed an increase in the levels of Th2-dependent cytokines and IgE, thereby reducing the symptoms of pollinosis. In the present study, to clarify the mechanism of action of adrenocorticotropic hormone (ACTH) in improving the symptoms of pollinosis, we studied the effects of ACTH on the plasma level of histamine, mast cell number in nasal-associated lymphoid tissue (NALT) and the T cell differentiation in splenocytes. METHODS: The role of ACTH in the development of pollen antigen-induced pollinosis was studied in mice. Allergic symptoms and parameters were measured on day 17 after sensitization. To investigate the effects of ACTH on T cell differentiation, we stimulated splenocytes obtained from control mice with ACTH and CD3/CD28 in vitro, and measured the cytokine production in the culture supernatant. RESULTS: The plasma levels of IL-10, IgE and histamine and mast cell number in NALT were increased in the sensitized animals in association with a concomitant increase in the incidence of sneezing and nasal rubbing. The intraperitoneal administration of ACTH decreased the IL-10, IgE and histamine levels in the plasma and mast cell number in NALT, while increasing the IFN-γ level and suppressing the incidence of nasal rubbing. Furthermore, the production of IFN-γ increased, while the IL-4 level was suppressed after 2 days in culture. CONCLUSIONS: The present findings showed that ACTH directly affects T cell differentiation and promotes Th1-type reactions. The regulation of the Th1/Th2 balance by ACTH may result in a decrease in the pathological features of pollinosis.

Stress-Induced Glucocorticoid Release Upregulates Uncoupling Protein-2 Expression and Enhances Resistance to Endotoxin-Induced Lethality.

OBJECTIVE: Although psychological and/or physiological stress has been well documented to influence immune responses, the precise mechanism for immunomodulation remains to be elucidated. The present work describes the role of the hypothalamic-pituitary-adrenal (HPA) axis in the mechanism of stress-mediated enhanced-resistance to lethality after lipopolysaccharide (LPS) injection. METHODS/RESULTS: Preconditioning with restraint stress (RS) resulted in enhanced activation of the HPA axis in response to LPS injection and suppressed LPS-induced release of proinflammatory cytokines and nitric oxide metabolites. Melanocortin 2 receptor-deficient mice (MC2R(-/-)) failed to increase plasma levels of glucocorticoids in response to LPS injection, and exhibited high sensitivity to LPS-induced lethality with enhanced release of proinflammatory cytokines as compared with MC2R(+/-) mice. Real-time PCR analysis revealed that RS induced upregulation of uncoupling protein-2 (UCP2) in macrophages in the lung and the liver of MC2R(+/-), but not of MC2R(-/-), mice. In addition, RS increased UCP2-dependent uncoupling activity of isolated mitochondria from the liver of MC2R(+/-), but not of MC2R(-/-), mice. In vitro study revealed that corticosterone and dexamethasone directly increased UCP2 expression in mouse RAW 264.7 macrophages and suppressed the generation of LPS-induced mitochondrial reactive oxygen species (ROS) and TNF-α production. Knockdown of UCP2 by small interfering RNA blunted the dexamethasone action for suppressing LPS-induced mitochondrial ROS and TNF-α production. CONCLUSION: The present work suggests that RS enhances activation of the HPA axis to release glucocorticoids and upregulation of UCP2 in macrophages, thereby increasing the resistance to endotoxin-induced systemic inflammation and death.

UVA irradiation of the eye modulates the contact hypersensitivity of the skin and intestines by affecting mast cells in mice.

BACKGROUND: Ultraviolet A (UVA) irradiation before allergic sensitization induces immunosuppression, but the precise mechanism remained unclear. In this study, we examined the influence of UVA irradiation of the eye on contact hypersensitivity (CHS) and the role of mast cells in CHS. METHODS: We used two types of haptens, fluorescein isothiocyanate (FITC: a Th2 type hapten) and 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone: a Th1 type hapten). A 300 kJ/m(2) dose of UVA irradiation was delivered to the eyes. After UVA irradiation, we sensitized abdominal shaved skin and challenged the ear epidermis and colons of these mice with each hapten. RESULTS: After UVA irradiation, the CHS of the skin and colon were not inhibited in the FITC-sensitized mice. However, in the oxazolone-sensitized mice, only the CHS of the skin was inhibited by UVA irradiation. The inflammation of the colon became more severe after UVA irradiation. In mast cell-deficient (W/Wv) mice sensitized to FITC, the CHS was weaker than that in WT mice. Moreover, the reduction of immunosuppression in ear swelling was seen for one of the two models they used. CONCLUSIONS: These results suggest that the mast cells induced by UVA irradiation of the eye have different roles in the epidermis and colon and have different responses to different haptens.

Isolation of diploid baker's yeast capable of strongly activating immune cells and analyses of the cell wall structure.

Diploid baker's yeast capable of strongly activating a mouse macrophage was constructed based on haploid mutant AQ-37 obtained previously. The obtained strain BQ-55 activated also human immune cells. To clarify a factor for the activation, the cell wall structure, especially the ß-glucan structure, was analyzed, suggesting that the length of branching, ß-1,6-glucan, may be one of the factors.

Gender difference in tumor necrosis factor-α production in human neutrophils stimulated by lipopolysaccharide and interferon-γ.

The gender difference in tumor necrosis factor-α (TNF-α) production in human neutrophils stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) was explored by using peripheral blood neutrophils from young men and women. As compared with female neutrophils, male neutrophils released greater amounts of TNF-α, and exhibited stronger activation of mitogen-activated protein kinases and phosphatidylinositol 3-kinase in response to LPS stimulation. LPS-induced TNF-α production was markedly enhanced by pretreatment of cells with IFN-γ, and IFN-γ-mediated priming in male neutrophils was significantly greater than that in female neutrophils. Male neutrophils showed higher expression of TLR4, but not IFN-γ receptors, than female neutrophils, and its expression was increased by stimulation with IFN-γ or IFN-γ plus LPS. These findings indicate that male neutrophils show higher responsiveness to stimulation with LPS and IFN-γ than female neutrophils, and suggest that the gender difference in neutrophil responsiveness to LPS and IFN-γ is partly responsible for that in the outcome of sepsis, in which premenopausal women show a favorable prognosis as compared with men.

Potent immunomodulating effects of bran extracts of traditional Japanese millets on nitric oxide and cytokine production of macrophages (RAW264.7) induced by lipopolysaccharide.

To estimate the potent immunomodulating effects of different types of traditional Japanese millet, we analyzed the effect of bran extracts of foxtail millet (Awa in Japanese), barnyard millet (Hie) and proso millet (Mochi-kibi) on nitric oxide (NO) and inflammatory cytokine production induced by lipopolysaccharide (LPS) in a mouse macrophage cell line (RAW264.7 cells). All methanol extracts of these millet brans showed suppressive activities against the production of NO and inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 in LPS-stimulated macrophages, which were not responsible for their cytotoxic activities. These immunosuppressive activities were roughly proportional to the contents of the phenolic compounds in their extracts. Especially, the extract of proso millet exhibited the strongest immunosuppressing effect, which was associated with the highest content of phenolic compound. However, the extracts did not exhibit significant suppressive effects on the production of an anti-inflammatory cytokine, IL-10, in the same macrophage culture system. These results suggest that traditional Japanese millets have potent immunomodulating activities against the production of NO and cytokine production in activated macrophages.

Mitochondria determine the efficacy of anticancer agents that interact with DNA but not the cytoskeleton.

Although chemotherapy is an important method for the treatment of patients with cancer, its efficacy is limited because of different sensitivities of tumor cells to anticancer agents and/or side effects on normal tissues. The present work demonstrates that mitochondria play a crucial role in the apoptosis of cancer cells induced by anticancer agents that interact with DNA but not with the cytoskeleton. Agents that interact with DNA selectively enhanced generation of reactive oxygen species (ROS) in mitochondria, released cytochrome c, and activated caspase-9 and caspase-3 to induce apoptosis of mesothelioma H2052 cells but not their ρ(0) cells, which lack mitochondrial DNA (mtDNA). The sensitivity of a variety of cells to the agents showed positive correlation with the amounts of their mitochondria. In contrast, agents that selectively affect the cytoskeleton activated caspase-8 and caspase-3 and equally induced apoptosis of both H2052 and their ρ(0) cells by a mitochondria-independent mechanism. The results suggest that mtDNA is a potential target for the anticancer agents that interact with DNA to induce ROS-dependent apoptosis of cancer cells, whereas agents that affect the cytoskeleton induce cell death by a mitochondria- and ROS-independent mechanism. The present observation is important for the selection of medicine for chemotherapy of patients with cancer.

Effects of Importin α1/KPNA1 deletion and adolescent social isolation stress on psychiatric disorder-associated behaviors in mice.

Importin α1/KPNA1 is a member of the Importin α family widely present in the mammalian brain and has been characterized as a regulator of neuronal differentiation, synaptic functionality, and anxiety-like behavior. In humans, a de novo mutation of the KPNA1 (human Importin α5) gene has been linked with schizophrenia; however, the precise roles of KPNA1 in disorder-related behaviors are still unknown. Moreover, as recent studies have highlighted the importance of gene-environment interactions in the development of psychiatric disorders, we investigated the effects of Kpna1 deletion and social isolation stress, a paradigm that models social stress factors found in human patients, on psychiatric disorder-related behaviors in mice. Through assessment in a behavioral battery, we found that Kpna1 knockout resulted in the following behavioral phenotype: (1) decreased anxiety-like behavior in an elevated plus maze test, (2) short term memory deficits in novel object recognition test (3) impaired sensorimotor gating in a prepulse inhibition test. Importantly, exposure to social isolation stress resulted in additional behavioral abnormalities where isolated Kpna1 knockout mice exhibited: (1) impaired aversive learning and/or memory in the inhibitory avoidance test, as well as (2) increased depression-like behavior in the forced swim test. Furthermore, we investigated whether mice showed alterations in plasma levels of stress-associated signal molecules (corticosterone, cytokines, hormones, receptors), and found that Kpna1 knockout significantly altered levels of corticosterone and LIX (CXCL5). Moreover, significant decreases in the level of prolactin were found in all groups except for group-housed wild type mice. Our findings demonstrate that Kpna1 deletion can trigger widespread behavioral abnormalities associated with psychiatric disorders, some of which were further exacerbated by exposure to adolescent social isolation. The use of Kpna1 knockout mice as a model for psychiatric disorders may show promise for further investigation of gene-environment interactions involved in the pathogenesis of psychiatric disorders.

Prevention of scattered light-induced asthenopia and fatigue by a polarized filter.

BACKGROUND: It has been well documented that a long-time irradiation of the eye by a strong light elicits eyestrain and fatigue. To elucidate the mechanism for the induction of light-induced fatigue and asthenopia, changes in the mouse were analyzed after white light-irradiation to the eye. METHODS: C57BL/6j male mice were irradiated with white light in a specially designed room equipped with four mirrors covering all areas of its four walls to elicit diffused reflected light, and changes in their plasma levels of cortisol, INF-gamma, interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) were analyzed. RESULTS: Irradiation of mice with scattered white light significantly decreased the motional activity of animals, suggesting the occurrence of fatigue. Biochemical analysis and enzyme-immunoassay revealed that the irradiation of mice significantly elevated the plasma levels of cortisol, IFN-gamma, IL-10 and TGF-beta. All these changes were not observed with mice irradiated with the light in a similar room not equipped with mirrors. These changes were successfully inhibited by a polarized glass filter but not by a non-polarized filter with a similar absorbance. CONCLUSIONS: These observations suggest that irradiation of the eye by scattered reflected light stimulated a stress response via hypothalamo-pituitary-adrenal axis to enhance the secretion of cortisol from the adrenal grand and increase the plasma levels of cytokines.

Dynamic aspects of ascorbic acid metabolism in the circulation: analysis by ascorbate oxidase with a prolonged in vivo half-life.

Because AA (L-ascorbic acid) scavenges various types of free radicals to form MDAA (monodehydroascorbic acid) and DAA (dehydroascorbic acid), its regeneration from the oxidized metabolites is critically important for humans and other animals that lack the ability to synthesize this antioxidant. To study the dynamic aspects of AA metabolism in the circulation, a long acting AOase (ascorbate oxidase) derivative was synthesized by covalently linking PEG [poly(ethylene glycol)] to the enzyme. Fairly low concentrations of the modified enzyme (PEG-AOase) rapidly decreased AA levels in isolated fresh plasma and blood samples with a concomitant increase in their levels of MDAA and DAA. In contrast, relatively high doses of PEG-AOase were required to decrease the circulating plasma AA levels of both normal rats and ODS (osteogenic disorder Shionogi) rats that lack the ability to synthesize AA. Administration of 50 units of PEG-AOase/kg of body weight rapidly decreased AA levels in plasma and the kidney without affecting the levels in other tissues, such as the liver, brain, lung, adrenal grand and skeletal muscles. PEG-AOase slightly, but significantly, decreased glutathione (GSH) levels in the liver without affecting those in other tissues. Suppression of hepatic synthesis of GSH by administration of BSO [L-buthionin-(S,R)-sulfoximine] enhanced the PEG-AOase-induced decrease in plasma AA levels. These and other results suggest that the circulating AA is reductively regenerated from MDAA extremely rapidly and that hepatic GSH plays important roles in the regeneration of this antioxidant.

Role of the hypothalamo-pituitary-adrenal axis in the modulation of pollinosis induced by pollen antigens.

BACKGROUND: To clarify the mechanism of stress-induced modification of allergic diseases, we studied the effect of restraint stress on plasma levels of cytokines and the symptoms of pollinosis in mice. METHODS: The effects of restraint stress and the role of the hypothalamo-pituitary-adrenal axis (HPA-axis) in the development of pollen antigen-induced pollinosis were studied in control, hypophysectomized, adrenalectomized or ACTH-administered mice. Twenty days after sensitization, animals were subjected to mild restraint stress for 3 hours, and plasma levels of IFN-gamma, IL-10, and IgE were measured. We analyzed the incidence of sneezing and nasal rubbing in the sensitized animals. RESULTS: Plasma levels of IL-10 and IgE increased in the sensitized animals with a concomitant increase in the incidence of sneezing and nasal rubbing. The increases in plasma IgE, IL-10 and the incidence of sneezing and nasal rubbing were suppressed by restraint stress. Adrenalectomy increased IFN-gamma, inhibited the increase in plasma IL-10 and IgE, and suppressed the incidence of sneezing. In contrast, hypophysectomy increased plasma levels of IL-10, IFN-gamma, and IgE and the incidence of sneezing. Intraperitoneal administration of ACTH decreased IL-10 in plasma but increased IFN-gamma and suppressed the incidence of nasal rubbing. CONCLUSIONS: The present findings show that the HPA-axis and ACTH play important roles in the regulation of plasma cytokines and IgE thereby modulating symptoms of pollinosis. The results also suggest that a mild restraint stress suppresses the increase in Th2-dependent cytokines and IgE to reduce the symptoms of pollinosis.

Decreased Colonic Guanylin/Uroguanylin Expression and Dried Stool Property in Mice With Social Defeat Stress.

Psychological stress is deeply involved in the pathophysiology of not only mental illness but also functional gastrointestinal disorders. In the present study, we examined the relationship between psychological stress and abnormality of stool properties, focusing on the alteration of plasma glucocorticoid and guanylin (GN)/uroguanylin (UGN) expression in the colon. A murine model of chronic social defeat stress (CSDS) was established by exposing a C57BL/6N intruder mouse to a CD-1 aggressor mouse for 3-5 min. Thereafter the mice were kept in the same cage but separated by a divider for the remainder of the day. This procedure was repeated for 10 consecutive days, and then a social interaction test was performed to evaluate social avoidance. Fresh fecal and blood samples were collected for stool property analysis and measurement of the plasma glucocorticoid level by ELISA. The expression of GN, UGN, and guanylate cyclase 2C in the colonic tissues was examined by real-time RT-PCR and immunohistochemistry. Moreover, Lovo cells were stimulated with dexamethasone, and the expression of GN/UGN mRNA was examined. In the CSDS group, the time spent in the social interaction zone was significantly shorter when the CD-1 aggressor mouse was present than when it was absent. The social interaction ratio was also significantly lower in the CSDS group relative to the controls. The mean Bristol scale score was significantly lower in the CSDS group, but the fecal sodium concentration did not differ between CSDS mice and controls. The level of plasma corticosterone was significantly higher in the CSDS group than in the controls immediately after the 10th day of CSDS. The expression of both GN and UGN was significantly decreased in the CSDS mice. GN was expressed in all colonic epithelial cells, and UGN was expressed in ovoid or pyramidal epithelial cells in the colonic mucosa. The expression of both GN and UGN was significantly decreased in the CSDS mice relative to controls. The expression of both GN and UGN was significantly suppressed in Lovo cells upon stimulation with dexamethasone. Psychological stress-induced glucocorticoid may suppress colonic GN/UGN expression, resulting in a change in stool properties leading to constipation.

Immunomodulating activities in bran extracts of Japanese red, black and brown rices.

To determine the potent immunomodulating activities of different types of Japanese rice bran, we analyzed the effects of extracts of red, brown and black rice brans on the cell proliferation and cytokine production of mouse immunocompetent cells by cell culture experiments. Methanol extracts of these rice brans showed suppressive activities against the proliferative response (3H-thymidine incorporation) of mouse spleen cells induced by concanavalin A (Con A) or lipopolysaccharide (LPS) in the cell culture experiments. Although the black and brown rice bran extracts showed suppressive effects on the production of interferon gamma (IFN gamma) or interleukin 6 (IL-6) in mouse spleen cells induced by Con A or LPS, the red rice bran extract exhibited stimulatory effects on the same cytokine-producing systems. Furthermore, when the effects of these extracts on the production of macropahage-derived inflammatory cytokines such as interleukin-1alpha (IL-1alpha) and tumor necrosis factor alpha (TNF-alpha) were assayed, the red rice bran extract caused a stimulatory effect on the IL-1alpha production from mouse macrophages induced by LPS, but did not show a significant effect on TNF-alpha production. However, the brown and black rice bran extracts exhibited significant inhibitory effects on the production of IL-1alpha and TNF-alpha in the same macrophage culture experiment. A possible mechanism of the immunomodulating activities of the rice bran extracts and the immunopharmacological significance of these findings are discussed.

Effect of Nitric Oxide on the Oxygen Metabolism and Growth of E. faecalis.

Gastro-intestinal mucosal cells have a potent mechanism to eliminate a variety of pathogens using enzymes that generate reactive oxygen species and/or nitric oxide (NO). However, a large number of bacteria survive in the intestine of human subjects. Enterococcus faecalis (E. faecalis) is a Gram-positive bacterium that survives not only in the intestinal lumen but also within macrophages generating NO. It has been reported that E. faecalis generated the superoxide radical (O(2) (-)). To elucidate the role of O(2) (-) and NO in the mechanism for the pathogen surviving in the intestine and macrophages, we studied the role and metabolism of O(2) (-) and NO in and around E. faecalis. Kinetic analysis revealed that E. faecalis generated 0.5 micromol O(2) (-)/min/10(8) cells in a glucose-dependent manner as determined using the cytochrome c reduction method. The presence of NOC12, an NO donor, strongly inhibited the growth of E. faecalis without affecting in the oxygen consumption. However, the growth rate of NOC12-pretreated E. faecalis in NO-free medium was similar to that of untreated cells. Western blotting analysis revealed that the NOC12-treated E. faecalis revealed a large amount of nitrotyrosine-posititive proteins; the amounts of the modified proteins were higher in cytosol than in membranes. These observations suggested that O(2) (-) generated by E. faecalis reacted with NO to form peroxinitrite (ONOO(-)) that preferentially nitrated tyrosyl residues in cytosolic proteins, thereby reversibly inhibited cellular growth. Since E. faecalis survives even within macrophages expressing NO synthase, similar metabolism of O(2) (-) and NO may occur in and around phagocytized macrophages.

An Inhibitor of Casein Kinase 1ε/δ (PF670462) Prevents the Deterioration of Dextran Sodium Sulfate-induced Ulcerative Colitis Caused by UVB Eye Irradiation.

Although we previously reported the exacerbation of dextran sodium sulfate (DSS)-induced ulcerative colitis by ultraviolet (UV) B eye irradiation, we do not yet understand the mechanism behind this phenomenon. In this study, we examined the relationship between the deterioration of DSS-induced ulcerative colitis and clock genes. We induced a mouse model of ulcerative colitis by administering DSS for 5 days, and administered UVB eye irradiation on each day of the DSS treatment period. The DSS-induced ulcerative colitis was deteriorated by the UVB eye irradiation. The levels of Clock, brain and muscle arnt-like protein 1 (Bmal1), reverse orientation c-erb A gene (Rev-Erb)α, RAR-related orphan receptor gamma (RORγt), and interleukin (IL)-17 in the colon were increased by UVB eye irradiation in the DSS-treated mice (UVB/DSS-treated mice). Conversely, the nuclear factor, interleukin 3 regulated (NFIL-3) levels in the colon were lower after UVB eye irradiation. The Casein Kinase 1ε/δ inhibitor (PF670462) administration, which is a Clock/Bmal1 inhibitor (PER2 activator), inhibited the deterioration caused by UVB eye irradiation. These results suggest that the UVB eye irradiation-mediated exacerbation of DSS-induced ulcerative colitis depends on IL-17 produced in response to alterations in clock genes.

The Clock Genes Are Involved in The Deterioration of Atopic Dermatitis after Day-and-Night Reversed Physical Stress in NC/Nga Mice.

BACKGROUND: In modern society, irregular lifestyles are a problem. It is well known that Atopic Dermatitis (AD) occurs during physical stress in people with an irregular lifestyle. We evaluated the influence that day-and-night reversal physical stress has on AD. METHODS: Six-week-old specific-pathogen-free and conventional NC/Nga male mice were used. For the day-and-night reversal procedure, the mice ran on a treadmill at a slow speed of 10 m/min for 12 h (between 8:00 and 20:00). Then, between 20:00 and 8:00, we put the mice in a dark place. This treatment was repeated every day for two weeks. The behavioral circadian rhythm of the mice was evaluated with the open field test. Then, the mice were sacrificed and histological examinations of the tissues, the expression of peptide hormones, corticosterone, Immunoglobulin E, histamine, and cytokines was performed using an enzyme-linked immunosorbent assay. RESULTS: In the treadmill-treated conventional NC/Nga mice, AD symptoms were deteriorated compared with the non-treated conventional NC/Nga mice. The levels of Period (Per) 2, Clock, and brain and muscle arnt-like protein 1 (Bmal1) in the skin were increased constantly in the treadmill-treated conventional mice. Furthermore, the expression of Retinoic Acid-related Orphan Receptor (ROR)α, which activates Bmal1, was increased in the treadmill-treated conventional mice compared with the non-treated conventional mice. In addition, when non-treated conventional mice were administrated by the agonist of RORα, AD symptoms were deteriorated similar to treadmill-treated conventional mice. CONCLUSION: In the day-and-night reversal mice, the clock genes were increased constantly, indicating that this is a factor that deteriorated AD.

Smoking a single cigarette rapidly reduces combined concentrations of nitrate and nitrite and concentrations of antioxidants in plasma.

BACKGROUND: Cigarette smoking is a well-known risk factor for the development of cardiovascular disease, yet the mechanism of action involved is not completely understood. Because cigarette smoke contains superoxide and other reactive oxygen species, it has been hypothesized that some of the adverse effects of smoking may result from oxidative damage to endothelial cells, which results in nitric oxide (NO) shortage. However, little information is available regarding the acute effects of smoking on plasma concentrations of NO and antioxidants. We measured the changes in the combined plasma concentrations of nitrate and nitrite as an index of NO concentration, as well as changes in concentrations of major serum antioxidants (ascorbic acid, cysteine, methionine, and uric acid) in smokers after smoking a single cigarette. METHODS AND RESULTS: A randomized crossover study of the effects of smoking a single cigarette was performed in 20 smokers. Smoking a sham cigarette induced no significant changes in all assayed parameters. However, smoking a single cigarette significantly decreased nitrate and nitrite plasma concentrations by 3.5 +/-1.2 and 3.4 +/- 1.1 micromol/L, compared with plasma concentrations at presmoking and sham smoking, respectively. The concentrations of ascorbic acid and other antioxidants were also significantly lower after smoking a single cigarette. These parameters returned to preexperimental levels 60 minutes after smoking cessation. CONCLUSION: The present findings indicate that smoking a single cigarette temporarily decreases nitrate, nitrite, and serum antioxidant concentrations in the plasma. These transient changes may partially contribute to coronary vasoconstriction, which is routinely observed after smoking.

SOD2 protects against oxidation-induced apoptosis in mouse retinal pigment epithelium: implications for age-related macular degeneration.

PURPOSE: Oxidative stress from reactive oxygen species (ROS) has been implicated in many diseases, including age-related macular degeneration (AMD), in which the retinal pigment epithelium (RPE) is considered a primary target. Because manganese superoxide dismutase (SOD2), localized in mitochondria, is known to be a key enzyme that protects the cells against oxidative stress, this study was undertaken to examine oxidation-induced apoptosis in cultured RPE cells with various levels of SOD2. METHODS: Primary cultures of RPE cells were established from wild-type (WT), heterozygous Sod2-knockout mouse (HET) and hemizygous Sod2 mice with overexpression of the enzyme (HEMI). Purity of the RPE cell cultures was verified by immunostaining with antibody to RPE65 and quantified by flow cytometry. Oxidative stress was induced in RPE cells by exposing them to H(2)O(2) (0-500 muM) for 1 hour and reculturing them in normal medium for various times (0-24 hours). Apoptosis in the RPE was examined by TUNEL staining and quantified by cell-death-detection ELISA. Mitochondrial transmembrane potential (MTP) was measured by a cationic dye, and cytochrome c leakage from mitochondria was analyzed by Western blot analysis. RESULTS: More than 95% of the cells in each culture were RPE65 positive, and the relative SOD2 levels in HET, WT, and HEMI cells were 0.6, 1.0, and 3.4, respectively. H(2)O(2)-induced apoptotic cell death was both dose and time dependent, and apoptosis in these cells was related to the cellular SOD2 level. Disruption of MTP and release of cytochrome c were observed to occur before apoptotic cell death, and they correlated with cellular SOD2. CONCLUSIONS: The results demonstrate a critical role of SOD2 in protection against oxidative challenge. Cells from HET mice showed greater apoptotic cell death, whereas in those from HEMI mice, cell death induced by oxidative injury was suppressed.