Sulfur-to-nitrogen transnitrosation: transfer of nitric oxide from S-nitroso compounds to diethanolamine and the role of intermediate sulfur-to-sulfur transnitrosation

S-Nitrosothiols are formed in vivo and are involved in NO signaling. We investigated the sulfur-to-nitrogen transnitrosation activity of S-nitrosocysteine, S-nitrosoglutathione, S-nitrosohomocysteine, S-nitrosocysteinylglycine and S-nitroso-N-acetylcysteine in their reaction with the secondary amine diethanolamine in vitro. The resulting N-nitrosodiethanolamine, a strong carcinogen, was formed in yields of up to 11% from S-nitrosocysteine and S-nitrosocysteinylglycine, whereas the transnitrosation activity of the other S-nitroso compounds was weak. However, the addition of L-cysteine to a solution of S-nitrosohomocysteine and diethanolamine accelerated the decomposition of S-nitrosohomocysteine and resulted in a significant formation of N-nitrosodiethanolamine accompanied by the intermediate generation of S-nitrosocysteine. Thus, reactive nitrosothiols can be formed from less reactive analogs via sulfur-to-sulfur transnitrosation. We suggest that this affects regulation of NO trafficking in vivo. The reaction provides an alternative mechanism for the generation of carcinogenic N-nitroso derivatives.     

High efficiency of 5-aminolevulinate-photodynamic treatment using UVA irradiation

Photodynamic therapy (PDT) is being used clinically for the treatment of skin cancers. One concept of delivering the employed photosensitizer directly to target cells is to stimulate cellular synthesis of sensitizers such as porphyrins. ALA (5-aminolevulinate) is applied as a precursor of porphyrins which then serve as endogenous photosensitizers. Upon irradiation, reactive oxygen species, predominantly singlet oxygen, are generated, leading to cell death. ALA-PDT using red light (550-750 nm) is known to lead to the activation of stress kinases, such as c-Jun-N-terminal kinase and p38. These kinases are also activated by UVA (320-400 nm), whose biological effects are mediated in part by singlet oxygen. In the present study, the efficiency of a combination of both treatment strategies, ALA-PDT and UVA, in cytotoxicity and activation of stress kinases was investigated taking human skin fibroblasts as a model. Compared with the commonly used ALA-PDT with red light (LD(50) = 13.5 J/cm(2)), UVA-ALA-PDT was 40-fold more potent in killing cultured human skin fibroblasts (LD(50) = 0.35 J/cm(2)) and still 10-fold more potent than ALA-PDT with green light (LD(50) = 4.5 J/cm(2)). Its toxicity relied on the formation of singlet oxygen, as was shown employing modulators of singlet oxygen lifetime. In line with these data, strong activation of the stress kinase p38 was obtained in ALA-pretreated cells irradiated with UVA at doses two orders of magnitude lower than necessary for a comparable activation of p38 by UVA in control cells. Taken together, these data suggest UVA-ALA-PDT as a potentially interesting new approach in the photodynamic treatment of skin diseases.     

Ebselen: prospective therapy for cerebral ischaemia

Stroke occurs due to haemorrhage or occlusive injury and results in ischaemia and reperfusion injury. A variety of destructive mechanisms are involved including oxygen radical generation, calcium overload, cytotoxicity and apoptosis as well as the generation of inflammatory mediators. Ebselen, 2-phenyl-1, 2-benzisoselenazol-3(2H)-one (PZ 51, DR3305), is a mimic of GSH peroxidase which also reacts with peroxynitrite and can inhibit enzymes such as lipoxygenases, NO synthases, NADPH oxidase, protein kinase C and H(+)/K(+)-ATPase. Ebselen is in a late stage of development for the treatment of stroke. The molecular actions of ebselen contribute to its anti-inflammatory and anti-oxidant properties, which have been demonstrated in a variety of in vivo models. Numerous in vitro experiments using isolated LDL, liposomes, microsomes, isolated cells and organs have established that ebselen protects against oxidative challenge. Unlike many inorganic and aliphatic selenium compounds, ebselen has low toxicity as metabolism of the compound does not liberate the selenium moiety, which remains within the ring structure. Subsequent metabolism involves methylation, glucuronidation and hydroxylation. Experimental studies in rats and dogs have revealed that ebselen is able to inhibit both vasospasm and tissue damage in stroke models, which correlates with its inhibitory effects on oxidative processes. Results from randomised, placebo-controlled, double-blind clinical studies on the neurological consequences of acute ischaemic stroke, subarachnoid haemorrhage and acute middle cerebral artery occlusion, have revealed that ebselen significantly enhances outcome in patients who have experienced occlusive cerebral ischaemia of limited duration. The benefit achieved with ebselen is closely related to the rapidity with which the treatment is initiated, following the onset of the stroke attack. Safety and tolerability are good and no adverse effects have become apparent. Ebselen is currently at the pre-registration stage for subarachnoid haemorrhage and stroke in Japan.     

Ca2+ mobilization by vasopressin and glucagon in perfused livers. Effect of prior intoxication with bromotrichloromethane

Perfused livers isolated from rats treated with BrCCl3 for up to 15 min were used as an experimental tool to investigate the role of the hepatic endoplasmic reticulum in Ca2+ mobilization elicited by vasopressin and glucagon. BrCCl3-treatment caused extensive impairment (37 to 92%) of Ca2+ pumps of isolated liver microsomes, while Ca2+ pumps of mitochondria and plasma membrane vesicles remained undamaged. In perfused livers of BrCCl3-treated rats, the efflux of Ca2+ and the concomitant stimulation of O2 consumption and glucose release induced by vasopressin were decreased. The extent of the decrease paralleled the duration of BrCCl3-treatment. The decrease of Ca2+ efflux following vasopressin addition was closely correlated with the decrease of active Ca2+ accumulation by isolated microsomes (r = 0.99, P less than 0.001). The Ca2+ efflux elicited by glucagon was also decreased after BrCCl3-treatment, whereas stimulation of O2 consumption and glucose release were retained. The possibility that BrCCl3-treatment might impair the production of the intracellular Ca2+-mobilizing messenger IP3 is unlikely, since vasopressin still induced the formation of inositol phosphates, including IP3, in isolated hepatocytes obtained from BrCCl3-treated rats. Thus, this work supports the hypothesis that the Ca2+ stored in the liver ER is the major pool of intracellular Ca2+ available for mobilization by vasopressin, glucagon and other effectors.     

Elevated lipid peroxidation biomarkers and low antioxidant status in atherosclerotic patients with increased carotid or iliofemoral intima media thickness.

OBJECTIVES: Lipid peroxidation plays an important role in the development of atherosclerosis, a chronic, age-related disease process of the arterial wall with onset decades prior to its clinical manifestations. The aim of the study was to assess the association between the intima media thickness (IMT) of the major arteries as a clinical marker of atherosclerosis and markers of lipid peroxidation along with the antioxidant status in humans. DESIGN: Case-control study. SETTING: A university-affiliated outpatient clinic. SUBJECTS: Thirty patients (22 males, 8 females; 70.4 +/- 7.3 years) with atherosclerosis of the carotid or iliofemoral arteries and 62 healthy controls (30 males, 32 females; 68.3 +/- 4.3 years). METHODS: Plasma levels of 8,12-isoprostane F2alpha-VI (8,12-IPF2alpha-VI) were measured by gas chromatography/mass spectrometry, whereas levels of malondialdehyde (MDA), vitamins A (retinol) and E (alpha- and gamma-tocopherol), and carotenoids were determined by high-performance liquid chromatography. The IMT was measured by B-mode ultrasonography. RESULTS: Patients showed, independent of fruit and vegetable intake, significantly lower plasma levels of retinol, alpha-tocopherol, and all carotenoids excluding beta-cryptoxanthin compared with controls. On the contrary, plasma 8,12-IPF2alpha-VI levels were almost doubled (p < .001) and MDA levels increased by one-third (p < .01) in atherosclerotic patients compared with controls. CONCLUSIONS: The analyses of isoprostanes and antioxidant nutrients in plasma as markers of oxidative stress and the parallel evaluation of IMT as a structural marker of atherosclerosis are suitable tools for investigating the role of antioxidants and oxidative stress in atherosclerosis.     

Intervention with flaxseed and borage oil supplements modulates skin condition in women

Ingestion of selected nutrients modulates dermal properties. In the present study, two groups of women ingested flaxseed or borage oil for 12 weeks. The control group received a placebo containing medium-chain fatty acids. Dose was 2.2 g total fatty acids/d with alpha-linolenic acid and linoleic acid as major constituents in the flaxseed oil group; in the borage oil group linoleic and gamma-linolenic acid were predominant. In the flaxseed oil group, the contribution of alpha-linolenic acid to total fatty acids in plasma was significantly increased on weeks 6 and 12, whereas there was an increase in gamma-linolenic acid in the borage oil group (P < 0.05). Skin irritation was performed by nicotinate treatment, and changes in skin reddening and blood flow were monitored. Compared to week 0, skin reddening was diminished in both groups; blood flow was also lowered. Skin hydration was significantly increased after 12 weeks of treatment compared to week 0, with flaxseed or borage oil (P < 0.05). Transepidermal water loss was decreased in both oil groups by about 10 % after 6 weeks of supplementation. A further decrease was determined after 12 weeks in the flaxseed oil group. Surface evaluation of living skin revealed that roughness and scaling of the skin were significantly decreased with flaxseed and borage oil comparing week 0 and week 12 (P < 0.05). Except for hydration, none of the parameters was affected in the placebo group. The present data provide evidence that skin properties can be modulated by an intervention with dietary lipids.     

NADPH-dependent oxidation of reduced ebselen, 2-selenylbenzanilide, and of 2-(methylseleno)benzanilide catalyzed by pig liver flavin-containing monooxygenase.

The selenazole ring-opened metabolites of ebselen, 2-selenylbenzanilide and 2-(methylseleno)benzanilide, are substrates for flavin-containing monooxygenase from pig liver. The Km values were 25 and 3 microM, respectively, measured at 37 degrees C, pH 7.4, in the presence of 1 mM GSH. The Vmax values were 390 mU/mg of protein, similar to those obtained with methimazole or other substrates for FMO1. Although ebselen also appears to be a substrate in the absence of GSH, it progressively inactivates the enzyme, apparently by binding covalently to essential enzyme thiols. The oxidation products of the selenol and methylseleno derivatives are rapidly reduced by GSH, regenerating the parent substrates. Rapid reduction of the selenide oxide by GSH was unexpected and suggests that, unlike S-oxidation of sulfides, Se-oxidation of selenides may be a route for bioactivation. The data show that in the presence of FMO1 micromolar amounts of either of these ring-opened metabolites establish a futile cycle catalyzing the oxidation of GSH to GSSG by NADPH and oxygen.     

Uptake of lycopene and its geometrical isomers is greater from heat-processed than from unprocessed tomato juice in humans.

Lycopene and beta-carotene are the most abundant carotenoids in human blood and tissues. Although lacking provitamin A activity, lycopene may be biologically active by contributing to the antioxidative defense system of the organism. We studied the uptake of lycopene from processed (boiled with 1% corn oil for 1 h) and unprocessed tomato juice in humans. Lycopene concentrations in human serum increased only when processed tomato juice was consumed. Lycopene uptake varied with individuals, but peak serum concentrations were always reached between 24 and 48 h. The carotenoid was eliminated from serum with a half-life of 2-3 d. The increase in peak serum concentrations was dose-dependent but not linear with the dose. Repeated doses led to a continual rise of lycopene in human serum. Of the different geometrical isomers (all-trans, 9-cis and 13-cis), the cis isomers seemed to be somewhat better absorbed than the all-trans form.     

Inhibition of superoxide and nitric oxide release and protection from reoxygenation injury by Ebselen in rat Kupffer cells.

Luminol chemiluminescence in phorbolester-activated cultured rat liver Kupffer cells was strongly inhibited by the selenoorganic compound ebselen (IC50 = 2 mumol/L). Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]one) also diminished reduction of ferricytochrome c (IC50 = 10 mumol/L), indicating a suppression of superoxide anion formation. Likewise, in lipopolysaccharide-pretreated Kupffer cells, ebselen proved to be a potent inhibitor of the conversion of oxyhemoglobin to methemoglobin (IC50 = 3 mumol/L) as a measure of nitric oxide formation. The sulfur-containing analog (2-phenyl-1,2-benzisothiazol-3[2H]one) and the ebselen derivative, methylselenobenzanilide, were inactive. These results indicate that ebselen is a potent inhibitor of NADPH oxidase in Kupffer cells, as has been reported for other macrophages and granulocytes. In addition, they suggest a novel characteristic of ebselen, namely very effective inhibition of nitric oxide synthase of macrophages. In line with its inhibitory effects on the release of reactive oxygen species by macrophages, complemented by its antioxidant properties, ebselen was potent in the prevention of reoxygenation injury of Kupffer cells (IC50 approximately 5 mumol/L).