Yno1p/Aim14p, a NADPH-oxidase ortholog, controls extramitochondrial reactive oxygen species generation, apoptosis, and actin cable formation in yeast.

The large protein superfamily of NADPH oxidases (NOX enzymes) is found in members of all eukaryotic kingdoms: animals, plants, fungi, and protists. The physiological functions of these NOX enzymes range from defense to specialized oxidative biosynthesis and to signaling. In filamentous fungi, NOX enzymes are involved in signaling cell differentiation, in particular in the formation of fruiting bodies. On the basis of bioinformatics analysis, until now it was believed that the genomes of unicellular fungi like Saccharomyces cerevisiae and Schizosaccharomyces pombe do not harbor genes coding for NOX enzymes. Nevertheless, the genome of S. cerevisiae contains nine ORFs showing sequence similarity to the catalytic subunits of mammalian NOX enzymes, only some of which have been functionally assigned as ferric reductases involved in iron ion transport. Here we show that one of the nine ORFs (YGL160W, AIM14) encodes a genuine NADPH oxidase, which is located in the endoplasmic reticulum (ER) and produces superoxide in a NADPH-dependent fashion. We renamed this ORF YNO1 (yeast NADPH oxidase 1). Overexpression of YNO1 causes YCA1-dependent apoptosis, whereas deletion of the gene makes cells less sensitive to apoptotic stimuli. Several independent lines of evidence point to regulation of the actin cytoskeleton by reactive oxygen species (ROS) produced by Yno1p.

Food-derived peroxidized fatty acids may trigger hepatic inflammation: a novel hypothesis to explain steatohepatitis.

BACKGROUND & AIMS: Obesity and hepatic steatosis are frequently associated with the development of a non-alcoholic steatohepatitis (NASH). The mechanisms driving progression of a non-inflamed steatosis to NASH are largely unknown. Here, we investigated whether ingestion of peroxidized lipids, as being present in Western style diet, triggers the development of hepatic inflammation. METHODS: Corn oil containing peroxidized fatty acids was administered to rats by gavage for 6 days. In a separate approach, hepatocytes (HC), endothelial (EC) and Kupffer cells (KC) were isolated from untreated livers, cultured, and incubated with peroxidized linoleic acid (LOOH; linoleic acid (LH) being the main fatty acid in corn oil). Samples obtained from in vivo and in vitro studies were mainly investigated by qRT-PCR and biochemical determinations of lipid peroxidation products. RESULTS: Rat treatment with peroxidized corn oil resulted in increased hepatic lipid peroxidation, upregulation of nitric oxide synthetase-2 (NOS-2), cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNFα), elevation of total nitric oxides, and increase in cd68-, cd163-, TNFα-, and/or COX-2 positive immune cells in the liver. When investigating liver cell types, LOOH elevated the secretion of TNFα, p38MAPK phosphorylation, and mRNA levels of NOS-2, COX-2, and TNFα, mainly in KC. The elevation of gene expression could be abrogated by inhibiting p38MAPK, which indicates that p38MAPK activation is involved in the pro-inflammatory effects of LOOH. CONCLUSIONS: These data show for the first time that ingestion of peroxidized fatty acids carries a considerable pro-inflammatory stimulus into the body which reaches the liver and may trigger the development of hepatic inflammation.

Antagonistic effects of selenium and lipid peroxides on growth control in early hepatocellular carcinoma.

UNLABELLED: Activation of the activator protein 1 (AP-1) transcription factor as well as increased serum levels of vascular endothelial growth factor (VEGF) and interleukin (IL)-8 predict poor prognosis of patients with hepatocellular carcinomas (HCCs). Moreover, HCC patients display reduced selenium levels, which may cause lipid peroxidation and oxidative stress because selenium is an essential component of antioxidative glutathione peroxidases (GPx). We hypothesized that selenium-lipid peroxide antagonism controls the above prognostic markers and tumor growth. (1) In human HCC cell lines (HCC-1.2, HCC-3, and SNU398) linoleic acid peroxide (LOOH) and other prooxidants enhanced the expression of VEGF and IL-8. LOOH up-regulated AP-1 activation. Selenium inhibited these effects. This inhibition was mediated by glutathione peroxidase 4 (GPx4), which preferentially degrades lipid peroxides. Selenium enhanced GPx4 expression and total GPx activity, while knock-down of GPx4 by small interfering RNA (siRNA) increased VEGF, and IL-8 expression. (2) These results were confirmed in a rat hepatocarcinogenesis model. Selenium treatment during tumor promotion increased hepatic GPx4 expression and reduced the expression of VEGF and of the AP-1 component c-fos as well as nodule growth. (3) In HCC patients, increased levels of LOOH-related antibodies (LOOH-Ab) were found, suggesting enhanced LOOH formation. LOOH-Ab correlated with serum VEGF and IL-8 and with AP-1 activation in HCC tissue. In contrast, selenium inversely correlated with VEGF, IL-8, and HCC size (the latter only for tumors smaller than 3 cm). CONCLUSION: Reduced selenium levels result in accumulation of lipid peroxides. This leads to enhanced AP-1 activation and consequently to elevated expression of VEGF and IL-8, which accelerate the growth of HCC. Selenium supplementation could be considered for investigation as a strategy for chemoprevention or additional therapy of early HCC in patients with low selenium levels.

Synthesis and characterization of 5-alkoxycarbonyl-4-hydroxymethyl-5-alkyl-pyrroline N-oxide derivatives.

The syntheses, analytical properties, and spin trapping behavior of four novel EMPO derivatives, namely 5-ethoxycarbonyl-4-hydroxymethyl-5-methyl-pyrroline N-oxide (EHMPO), 5-ethoxycarbonyl-5-ethyl-4-hydroxymethyl-pyrroline N-oxide (EEHPO), 4-hydroxymethyl-5-methyl-5-propoxycarbonyl-pyrroline N-oxide (HMPPO), and 4-hydroxymethyl-5-methyl-5-iso-propoxycarbonyl-pyrroline N-oxide (HMiPPO), towards different oxygen- and carbon-centered radicals are described.

Synthesis and characterization of 5-hydroxymethyl-5-methyl-pyrroline N-oxide and its derivatives.

Synthesis and spin trapping behavior of three novel DMPO derivatives, namely 5-hydroxymethyl-5-methyl-pyrroline N-oxide (HMMPO), 5-(2-furanyl)-oxymethyl-5-methyl-pyrroline N-oxide (FMMPO), and 5-(2-pyranyl)-oxymethyl-5-methyl-pyrroline N-oxide (PMMPO) towards different oxygen- and carbon-centered radicals are described. The stabilizing effect of a series of cyclodextrins on the superoxide adducts was tested.

Polyphenol Diversity and Antioxidant Activity of European Cistus creticus L. (Cistaceae) Compared to Six Further, Partly Sympatric Cistus Species.

This investigation focused on the qualitative and quantitative composition of polyphenolic compounds of Mediterranean northern shore Cistus creticus and six further, partly sympatric Cistus species (C. albidus, C. crispus, C. ladanifer, C. monspeliensis, C. parviflorus, C. salviifolius). Aqueous extracts of 1153 individual plants from 13 countries were analyzed via high performance liquid chromatography (HPLC). The extracts of C. creticus were primarily composed of two ellagitannins (punicalagin and punicalagin gallate) and nine flavonol glycosides (myricetin and quercetin glycosides, with m-3-O-rhamnoside as the dominant main compound). Differences in the proportions of punicalagin derivatives and flavonol glycosides allowed the classification into two chemovariants. Plants containing punicalagin derivatives and flavonol glycosides were especially abundant in the western and central Mediterranean areas and in Cyprus. From Albania eastwards, punicalagin and punicalagin gallate were of much lesser importance and the predominant chemovariant there was a nearly pure flavonol type. With its two chemovariants, C. creticus takes a central position between the flavonol-rich, purple-flowered clade (besides C. creticus, here represented by C. albidus and C. crispus) and the more ellagitannin-rich, white- or whitish-pink-flowered clade (here represented by C. ladanifer, C. monspeliensis, C. parviflorus and C. salviifolius). The median antioxidative capacity of C. creticus plant material was, with 166 mg Trolox equivalents/g dry wt, about half of the antioxidative capacity of C. ladanifer (301 mg te/g dry wt), the species with the highest antioxidative potential.

Activation of artemisinin and heme degradation in Leishmania tarentolae promastigotes: A possible link.

Endoperoxides (EPs) appear to be promising drug candidates against protozoal diseases, including malaria and leishmaniasis. Previous studies have shown that these drugs need an intracellular activation to exert their pharmacological potential. The efficiency of these drugs is linked to the extensive iron demand of these intracellular protozoal parasites. An essential step of the activation mechanism of these drugs is the formation of radicals in Leishmania. Iron is a known trigger for intracellular radical formation. However, the activation of EPs by low molecular iron or by heme iron may strongly depend on the structure of the EPs themselves. In this study, we focused on the activation of artemisinin (Art) in Leishmania tarentolae promastigotes (LtP) in comparison to reference compounds. Viability assays in different media in the presence of different iron sources (hemin/fetal calf serum) showed that IC50 values of Art in LtP were modulated by assay conditions, but overall were within the low micromolar range. Low temperature electron paramagnetic resonance (EPR) spectroscopy of LtP showed that Art shifted the redox state of the labile iron pool less than the EP ascaridole questioning its role as a major activator of Art in LtP. Based on the high reactivity of Art with hemin in previous biomimetic experiments, we focused on putative heme-metabolizing enzymes in Leishmania, which were so far not well described. Inhibitors of mammalian heme oxygenase (HO; tin and chromium mesoporphyrin) acted antagonistically to Art in LtP and boosted its IC50 value for several magnitudes. By inductively coupled plasma methods (ICP-OES, ICP-MS) we showed that these inhibitors do not block iron (heme) accumulation, but are taken up and act within LtP. These inhibitors blocked the conversion of hemin to bilirubin in LtP homogenates, suggesting that an HO-like enzyme activity in LtP exists. NADPH-dependent degradation of Art and hemin was highest in the small granule and microsomal fractions of LtP. Photometric measurements in the model Art/hemin demonstrated that hemin requires reduction to heme and that subsequently an Art/heme complex (λmax 474 nm) is formed. EPR spin-trapping in the system Art/hemin revealed that NADPH, ascorbate and cysteine are suitable reductants and finally activate Art to acyl-carbon centered radicals. These findings suggest that heme is a major activator of Art in LtP either via HO-like enzyme activities and/or chemical interaction of heme with Art.

Synthesis and characterization of several carbamoyl- and methylcarbamoyl-substituted EMPO derivatives.

The spin trapping behavior of four novel carbamoyl-substituted EMPO derivatives, namely 5-carbamoyl-3,5-dimethyl-pyrroline N-oxide (CADMPO), 3,5-dimethyl-5-methylcarbamoyl-pyrroline N-oxide (DMMCAPO), 5-carbamoyl-3-ethyl-5-methyl-pyrroline N-oxide (CAEMPO), and 3-ethyl-5-methyl-5-methylcarbamoyl-pyrroline N-oxide (EMMCAPO), towards different oxygen- and carbon-centered radicals is described, the half lives of the respective superoxide adducts ranging from about 10 to 20 min. The most characteristic adducts were, however, formed from methyl, hydroxymethyl, hydroxyethyl, and carbon dioxide anion radicals.

Spin trapping experiments with different carbamoyl-substituted EMPO derivatives.

The spin trapping behavior of five carbamoyl-substituted EMPO derivatives, 5-aminocarbonyl-5-methyl-pyrroline N-oxide (CAMPO (AMPO)), 5-aminocarbonyl-5-ethyl-pyrroline N-oxide (CAEPO), 5-aminocarbonyl-5-propyl-pyrroline N-oxide (CAPPO), 5-aminocarbonyl-5-n-butyl-pyrroline N-oxide (CABPO), and 5-aminocarbonyl-5-n-pentyl-pyrroline N-oxide (CAPtPO), toward different oxygen- and carbon-centered radicals is described, the stabilities of the superoxide adducts ranging from about 8 to 17min.

Impact of glutathione peroxidase 4 on cell proliferation, angiogenesis and cytokine production in hepatocellular carcinoma.

Insufficient supplementation with the micronutrient selenium and persistent hepatic inflammation predispose to hepatocellular carcinoma (HCC). Inflammation-associated reactive oxygen species attack membrane lipids and form lipid hydroperoxides able to propagate oxidative hepatic damage. Selenium-containing enzyme glutathione peroxidase 4 (GPx4) antagonizes this damage by reducing lipid hydroperoxides to respective hydroxides. However, the role of GPx4 in HCC remains elusive. We generated two human HCC cell lines with stable overexpression of GPx4, performed xenotransplantation into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) host mice and characterized the tumors formed. The experimental data were verified using gene expression data from two independent HCC patient cohorts. GPx4 overexpression protected from oxidative stress and reduced intracellular free radical level. GPx4-overexpressing cells displayed impaired tumor growth, reduced proliferation, altered angiogenesis and decreased expression of clinically relevant cytokine interleukin-8 and C-reactive protein. Moreover, GPx4 overexpression impaired migration of endothelial cells in vitro, and enhanced expression of thrombospondin 1, an endogenous inhibitor of angiogenesis. In patients, GPx4 expression in tumors positively correlated with survival and was linked to pathways which regulate cell proliferation, motility, tissue remodelling, immune response and M1 macrophage polarization. The patient data largely confirmed experimental findings especially in a subclass of poor prognosis tumors with high proliferation. GPx4 suppresses formation and progression of HCC by inhibition of angiogenesis and tumor cell proliferation as well as by immune-mediated mechanisms. Modification of GPx4 expression may represent a novel tool for HCC prevention or treatment.

Oxidative stress mediates an increased formation of vascular endothelial growth factor in human hepatocarcinoma cells exposed to erlotinib.

The tyrosine kinase inhibitor erlotinib targets the receptor of epidermal growth factor (EGFR) involved in development of hepatocellular carcinoma (HCC). Although inefficient in established HCC, erlotinib has been recently proposed for HCC chemoprevention. Since Cyp3A4 and Cyp1A2 enzymes metabolize erlotinib in the liver, the insights into the mechanisms of erlotinib effects on liver cells with maintained drug metabolizing activity are needed. We applied erlotinib to both commercially available (SNU398, Huh7) and established in Austria HCC cell lines (HCC-1.2, HCC-3). Cyp3A4 and Cyp1A2, microarray gene expression, cell viability, LDH release, DHFC fluorescence were assessed. VEGF expression was analysed by real-time RT-PCR and ELISA. Higher cumulative expression of erlotinib metabolizing enzymes was observed in HCC-1.2 and HCC-3 cells. Gene expression microarray analysis showed upregulation of VEGF signalling by erlotinib. VEGF was increased up to 134 ± 14% (n = 5, p = 0.002) in HCC-1.2, HCC-3 and Huh7 cells. Interventions by Cyp1A2 and Mek2siRNA, MEK inhibitor UO126, diphenylene iodonium, as well as a combination of N-acetylcysteine with selenium all inhibited VEGF upregulation caused by erlotinib. Thus, erlotinib increases VEGF production by mechanisms involving Cyp1A2, oxidative stress and MEK1/2. VEGF may favour angiogenesis and growth of early HCC tumours limiting the therapeutic and chemopreventive effects of erlotinib.

Are the biological properties of kaempferol determined by its oxidation products?

Although flavonoid molecules have attracted considerable interest in recent years because of their antioxidant effect, there are considerable differences in their chemical properties. Electron paramagnetic resonance (EPR) spectroscopy was used to compare the oxidative free radical chemistry of two such molecules, kaempferol and luteolin, which have the same empirical formula but differ in the position of one OH group. Whereas the basic flavonoid structure remains intact in luteolin, structural changes occur in kaempferol after one-electron oxidation. Autoxidation of kaempferol in alkaline solution and oxidation by at pH 7 led to rapid fragmentation. In contrast, oxidation by horseradish peroxidase/hydrogen peroxide, xanthine/xanthine oxidase (X/XO) or a Fenton reaction system produced a radical whose structure appeared to be based on dimerisation of either the original or a fragment of the flavonoid. Hence, the biological properties of kaempferol are likely to be determined by the chemistry of its oxidation products.

Spin adduct formation from lipophilic EMPO-derived spin traps with various oxygen- and carbon-centered radicals.

Free radicals are involved in the onset of many diseases, therefore the availability of adequate spin traps is crucial to the identification and localization of free radical formation in biological systems. In recent studies several hydrophilic compounds of 2-ethoxycarbonyl-2-methyl-pyrroline-N-oxide (EMPO) have been found to form rather stable superoxide spin adducts with half-lives up to twenty minutes at physiological pH. This is a major improvement over DMPO (t1/2=ca. 45 s), and even over DEPMPO (t1/2=ca. 14 min), the best commercially available spin trap for the unambiguous detection of superoxide radicals. In order to allow the detection of superoxide and also other radicals in lipid environment a series of more lipophilic derivatives of EMPO was synthesized and their structure unambiguously characterized by 1H and 13C NMR spectroscopy. In this way, six different compounds with a n-butyl group in position 5 and either an ethoxy- (EBPO), propoxy- (PBPO), iso-propoxy- (iPBPO), butoxy- (BBPO), sec-butoxy- (sBBPO) or tert-butoxycarbonyl group (tBBPO) in position 5 of the pyrroline ring were obtained and fully analytically characterized (NMR, IR). The stability of the superoxide adducts of all investigated spin traps were comparable with EMPO (t1/2=ca. 8 min), except for the two compounds bearing an additional methyl group in position 3 or 4 of the pyrroline ring, 5-butyl-5-ethoxycarbonyl-3-methyl-pyrroline-N-oxide (BEMPO-3) and 5-butyl-5-ethoxycarbonyl-4-methyl-pyrroline-N-oxide (BEMPO-4), of which the superoxide adducts were stable for more than 30 min. Spin adducts of other carbon- and oxygen-centered radicals were also investigated.

Very stable superoxide radical adducts of 5-ethoxycarbonyl- 3,5-dimethyl-pyrroline N-oxide (3,5-EDPO) and its derivatives.

Oxygen radicals are involved in the onset of many diseases. Adequate spin traps are required for identification and localisation of free radical formation in biological systems. Superoxide spin adducts with half-lives up to 20 min at physiological pH have recently been reported to be formed from derivatives of the spin trap 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO). This is a major improvement over DMPO (t(1/2) ca. 45 s), and even DEPMPO (t(1/2) ca. 14 min). In this study, an additional methyl group was introduced into position 3 or 4 of the pyrroline ring which greatly increases the stability of the respective superoxide spin adducts. In addition, the ethoxy group of EMPO was exchanged by either a propoxy- or an iso-propoxy group in order to test the influence of increasing lipophilic properties of the investigated spin traps. The structure of all compounds was confirmed by (1)H and (13)C-NMR with full signal assignment. In comparison with EMPO (t(1/2) ca. 8 min) or DEPMPO (t(1/2) ca. 14 min), the superoxide adducts of all novel spin traps were considerably higher (t(1/2) ca. 12-55 min). In addition, various other spin adducts obtained from oxygen-centered as well as carbon-centered radicals (e.g. derived from methanol or linoleic acid hydroperoxide) were also detected.

Effect of Copper on Fatty Acid Profiles in Non- and Semifermented Teas Analyzed by LC-MS-Based Nontargeted Screening.

Unsaturated fatty acids are well-known precursors of aroma compounds, which are considered important for green tea quality. Due to the known copper-induced oxidation of unsaturated fatty acids and the broad variability of the amount of copper present in tea infusions, this paper investigates the influence of copper, added at a nontoxic concentration (300 µM) to non- and semifermented teas, on the degradation of fatty acids and fatty acid hydroperoxides thereof. The abundance of fatty acids in green and oolong tea was determined by means of a nontargeted approach applying high-resolution MS/MS. As a result, most of the fatty acids in green and oolong tea were already oxidized prior to copper addition. Addition of 300 µM CuSO4 to the oolong tea sample resulted in a decrease of 13-hydroperoxy-9Z,11E-octadecadienoic acid, an important flavor precursor, from 0.12 ± 0.02 to 0.05 ± 0.01 µM (p = 0.035), and other oxidized fatty acids decreased as well. However, copper-induced degradation of oxidized fatty acids was less pronounced in green tea compared to oolong tea, most likely due to the formation of copper complexes with low-molecular-weight compounds as evidenced by electron paramagnetic resonance spectroscopy.

Investigations into cytotoxic effects of the herbal preparation Abnormal Savda Munziq.

OBJECTIVE: To evaluate the effects of Abnormal Savda Munziq (ASMq), a traditional herbal medicine, for the prevention and treatment of human diseases, e.g. bowel cancer. METHODS: The parameters total polyphenol content, cell proliferation and DNA-damage as well as RNA and protein-oxidation were analysed in vitro. Besides, the expressions of miRNA and tumor suppressor genes as well as cellular senescence were evaluated. RESULTS: ASMq had a high polyphenol content and induced damage to proteins, RNA as well as to DNA, which is correlated with its cytotoxicity. Furthermore ASMq up-regulated the tumor suppressor genes p21, p53 and p16 and down-regulated the micro-RNAs hsa-mir-17 and hsa-mir-106b. In addition cellular growth arrest and SA-ß-gal-staining were induced. CONCLUSION: ASMq has the ability to induce DNA damage and cellular senescence, which are double-edged mechanisms in fighting cancer, as they might also have harmful side effects.

ESR analysis of spin adducts of alkoxyl and lipid-derived radicals with the spin trap Trazon.

Detection of oxygen-centered radicals was performed using the spin trap 1,3,3-trimethyl-6-azabicyclo[3.2.1]oct-6-ene-N-oxide (Trazon), a bicyclic nitrone spin trap that is easily synthesized from the corresponding amine via hydrogen peroxide mediated oxidation in the presence of the catalyst, sodium tungstate. Compared to monocyclic spin traps such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), the ESR spectra of Trazon spin adducts provide additional structural information due to long-range hyperfine splitting constants and also due to the fact that different stereoisomers can be distinguished. This is especially helpful for the detection of lipid-derived alkoxyl radicals which can be identified according to their characteristic hyperfine splitting pattern. Due to the relatively high stability of the Trazon spin adducts with lipid alkoxyl radicals, which were formed from peroxidizing linoleic acid, ESR experiments could be performed using a stationary system, whereas a slow-flow system is recommended for DMPO. A series of structurally different alkoxyl radical adducts were synthesized by iron-catalyzed nucleophilic addition of the respective alcohol to the spin trap Trazon and the spectra were analyzed by computer simulation. Both the molecular weight of the alcohol and the position of the alcoholic hydroxyl group were of significant influence on the ESR spectra. Two stereochemically different spin adducts were formed in a ratio typical of the alcohol used, thus allowing structural classification of the alkoxyl radical trapped.

Spin trapping of superoxide, alkyl- and lipid-derived radicals with derivatives of the spin trap EPPN.

The N-t-butyl-alpha-phenylnitrone derivative N-2-(2-ethoxycarbonyl-propyl)-alpha-phenylnitrone (EPPN) has recently been reported to form a superoxide spin adduct (t(1/2)=5.25 min at pH 7.0), which is considerably more stable than the respective N-t-butyl-alpha-phenylnitrone or 5,5-dimethylpyrroline N-oxide adducts (t(1/2) approximately 10 and 45s, respectively). In continuation of our previous studies on structure optimization of 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide derivatives, a series of six different EPPN derivatives was synthesized and characterized by 1H NMR, 13C NMR and IR spectroscopy. The ethoxy group of EPPN was replaced by a propoxy, iso-propoxy, n-butoxy, sec-butoxy, and tert-butoxy moiety, as well as the phenyl by a pyridyl ring. Electron spin resonance spectra and stabilities of the superoxide adducts of the propoxy derivatives were found to be similar to those of the respective EPPN adduct, whereas the electron spin resonance spectra of the superoxide adducts of N-2-(2-ethoxycarbonyl-propyl)-alpha-(4-pyridyl) nitrone and the butoxy derivatives were accompanied by decomposition products. In contrast to the 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide series, no significant improvement of the superoxide adduct stability could be obtained when the ethoxy group was replaced by other substituents. Carbon centered radical adducts derived from methanol, ethanol, formic acid and linoleic acid hydroperoxide were more stable than those of 5,5-dimethylpyrroline N-oxide, whereas among the alkoxyl radicals only the methoxyl radical adduct could be detected.

Spin adducts of several N-2-(2-alkoxycarbonyl-propyl)-alpha-pyridylnitrone derivatives with superoxide, alkyl and lipid-derived radicals.

Several derivatives of N-t-butyl-alpha-phenylnitrone (PBN) such as N-2-(2-ethoxycarbonyl-propyl)-alpha-phenylnitrone (EPPN) have recently been reported to form superoxide spin adducts (t(1/2) ca. 2-7 min at pH 7.0), which are considerably more stable than their respective PBN or DMPO adducts (t(1/2) ca. 10 and 45 s, respectively). In continuation of our studies on structure optimization of EPPN derivatives, a series of 12 novel spin traps with 2-, 3- and 4-pyridinyl substituents was synthesized and fully characterized by 1H NMR, 13C NMR and IR spectroscopy. In addition to the replacement of the phenyl ring by a 2-, 3- or 4-pyridinyl substituent, the ethoxy group of the parent compound EPPN was replaced by either a propoxy, iso-propoxy, or cyclopropylmethoxy moiety. Superoxide adducts of all PPyN derivatives were considerably more stable than those of the respective EPPN derivatives with half-lives ranging from about 6 to 11 min. In addition, alkoxyl radical adducts were also considerably more stable than those of the EPPN series. Hydroxyl radical adducts were not detected, on the other hand, very stable spin adducts were formed from a series of carbon centered radicals, e.g. from the methyl or hydroxymethyl radical. The novel spin traps are offering an alternative to PBN or POBN, especially where the higher stability of oxygen-centered radical adducts is of major importance. All of them can easily be synthesized from commercially available compounds in two or three steps.

Reactive oxygen species are involved in the stimulation of the mitochondrial permeability transition by dihydrolipoate.

Dihydrolipoic acid (DHLA) has been found to stimulate the Ca(2+)-induced mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) [Biochem. Mol. Biol. Int. 44 (1998) 127] which could be due to its prooxidant properties. We therefore investigated whether DHLA stimulated superoxide anion (O(2)(.-)) generation in RLM and in bovine heart submitochondrial particles (SMP). In RLM DHLA caused a concentration-dependent O(2)(.-) generation assayed by lucigenin chemiluminiscence. The stimulation was seen with the lowest concentrations of DHLA (5 microM) with pyruvate as the respiratory substrate, with 2-oxoglutarate or especially succinate the stimulation was less pronounced. Stimulation of O(2)(.-) production by DHLA was also observed in bovine heart SMP using an electron spin-trapping technique. Radical scavengers (butylhydroxytoluene and TEMPO) decreased O(2)(.-) generation induced by DHLA and inhibited MPT. Slight reduction of the mitochondrial membrane potential by a small amount of a protonophorous uncoupling agent also delayed the DHLA-induced MPT. These data indicate that the stimulation of MPT by DHLA is due to DHLA-derived prooxidants, i.e. stimulated production of O(2)(.-) and possibly other free radicals.