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Physiological effects of the reactive sulphur species (RSS) allicin from garlic
Keywords: NAD(P)H oxidases, Hydrogen peroxide, Aquaporins, Leukaemia cells doi:10.1016/j.freeradbiomed.2012.08.332 [0511] The First Purification and Characterization of a Novel Carbon-centered Quinone Ketoxy Radical Adduct B-Z. Zhu*, C-H. Huang Research Centre for Eco-Environmental Sciences, Chinese Academy of Sciences, China Despite being detected as a novel radical intermediate during the reaction between halogenated quinones and hydroperoxides, the carbon-centered quinone ketoxy radical adduct with the spin-trapping agents have proven very challenging to isolate and characterize. Here we show that a relatively stable nitroxide adduct of the quinone ketoxy radical with a half-life about 5 hours could be formed with the new spin-trapping agent BMPO (5-t-butoxycarbonyl 5-methyl-1-pyrroline N-oxide), an analog of the well-known DMPO. Interestingly and unexpectedly, we can not only directly detect the BMPOquinone ketoxy nitroxide radical adduct by mass spectrometry, but also isolate and purify it by semipreparative HPLC, which enables direct observation of its pure 6-line signal by ESR without any interference of other concurrently-formed radicals. This study represents the first purification and identification of an unusual quinone ketoxy radical adduct through complementary application of ESR spin trapping and HPLC/MS methods, which provides strong and direct experimental evidence for the existence of such an unusual quinone radical during metal-independent decomposition of hydroperoxides and formation of alkoxyl/hydroxyl radicals by halogenated quinones. Keywords: Quinone ketoxy radical, radical purification by HPLC, radical identification by mass spectrometry, ESR spin-trapping doi:10.1016/j.freeradbiomed.2012.08.333 [0517] How Much Hydroxyl Radicals can be Produced from a Novel Metal-independent Hydroxyl RadicalGenerating System? B-Z. Zhu*, H-Y. Xia Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, China
We found recently that hydroxyl radical (•OH) could be produced by chlorinated quinones (CBQs) and hydrogen peroxide (H2O2) via a novel metal-independent nucleophilic substitution and homolytical decomposition mechanism. However, it is still not clear how much •OH can be produced by this unusual •OH generating system. In this work, the •OH yield by CBQs and H2O2 was quantitatively determined using salicylic acid (SA) hydroxylation method coupled with HPLCelectrochemical detection, and compared with that produced by the classic iron-mediated Fenton system (Fe(II)EDTA/H2O2). We found that •OH was produced both time- and dose-dependently on CBQs, H2O2 and SA as well as pH. Interestingly, the •OH production by CBQs/H2O2 was found to be maximal under weakly alkaline condition (pH, ≈ 8.0) while that by the classic Fenton system was maximal under weakly acidic condition (pH, ≈ 6.0) The maximal •OH yield by CBQs/H2O2 was found to be dependent on both the number and the position of the chlorine substitution. Surprisingly, some metal-independent •OH producing CBQs/H2O2 system was found to generate even more • OH than the classic Fenton system under the optimal conditions. In conclusion, CBQ/H2O2 system is a very efficient •OH producing system, and we suggest that they can be used to substitute the classic Fenton system to produce •OH for various biological studies, especially when there is a need to exclude the presence of transition metal ions. Keywords: Hydroxyl radical, chlorinated quinones, salicylic acid hydroxylation, 2,3-/2,5-dihydroxybenzoic acid doi:10.1016/j.freeradbiomed.2012.08.334 [0573] Physiological effects of the reactive sulphur species (RSS) allicin from garlic A.J. Slusarenko*, M.C.H. Gruhlke RWTH Aachen University, Germany Allicin (diallyl thiosulfinate) is a naturally occurring reactive sulphur species (RSS)1. Allicin is volatile, antimicrobial and is produced in garlic when the tissues are damaged; giving freshly cut garlic its characteristic odour. Allicin is readily membrane-permeable and a prooxidant which undergoes thiol-disulphide exchange reactions with free thiol groups in proteins. It is thought that these properties are the basis of its antimicrobial action. Allicin is part of the defence repertoire employed by garlic to defend itself against pathogens and pests. We have shown the effectiveness of pure synthetic allicin and garlic juice containing allicin, against a range 2 of plant pathogenic organisms in vitro and in planta . In
S159
addition to reacting with thiol groups in proteins we have shown that synthetic allicin induces apoptosis in yeast by altering the glutathione pool (GSH:GSSG) and disturbing the cell’s electrochemical potential3. We are currently characterizing the cellular details of the allicin-induced route into apoptosis in baker’s yeast (Saccharomyces cerevisiae) as a model fungus. Our results show that protein kinase A (PKA) activity is necessary for allicininduced apoptosis to occur and appears to coordinate apoptosis inducing factor (AIF) activity. Furthermore, we have shown that in a concentration-dependent manner, allicin permeabilizes cell membranes and is more active on a molar basis than dimethylsulfoxide (DMSO) in this respect. Thus, allicin not only has potential for development in plant protection, but it is also a useful RSS tool to investigate fungal apoptosis and various aspects of cell physiology. 1
Gruhlke and Slusarenko (2012) Plant Physiology and Biochemistry. http://dx.doi.org/10.1016/j.plaphy.2012.03.016 2 Curtis, et al. (2004) Physiological and Molecular Plant Pathology 65, 79-89 3 Gruhlke et al. (2010) Free Radical Biology and Medicine 49, 1916-1924, doi:10.1016/j.freeradbiomed.2010.09.019 Keywords: glutathione, species, allicin
apoptosis,
reactive
citrin deficiency there is a defect in the mitochondrial aspartate-glutamate carrier, leading to compensatory up regulation of the malate-citrate carrier, which causes an increase in the cytosolic citrate concentration. Interestingly, citrate is a metabolic intermediate that itself cannot be oxidized by the cell. We hypothesized that increased citrate levels, caused by an abundance of fatty acids, contribute to the development of NASH by an increase of oxidative stress. Therefore the aim of this study was to investigate citrate levels in NASH patients and to investigate the effect of citrate on oxidative stress. In line with our hypothesis, we found significantly increased blood plasma levels of citrate in NASH patients. Moreover, addition of palmitic acid to the incubation medium of immortalized hepatocytes (HepG2 cells) was found to increase the intracellular citrate concentration. This was measured with the use of liquid chromatography-tandem mass spectrometry. Oxidative stress in HepG2 cells exposed to citrate was measured with the use of 2'7'- dichlorofluorescein (DCFH). Citrate was found to increase radical formation by the Fenton reaction, which is probably related to the ability of citrate to chelate iron. Based on these findings, we postulate that abundance of fatty acids in NASH results in elevated citrate levels. This can lead to an increase of oxidative stress which contributes to the development of NASH.
sulfur
doi:10.1016/j.freeradbiomed.2012.08.335
Keywords: Non-alcoholic Oxidative stress, Fatty acids
steatohepatitis,
Citrate,
doi:10.1016/j.freeradbiomed.2012.08.336 [0585] [0588] Increase of oxidative stress in NASH by increased levels of citrate B. van de Wier*1, G.R.M.M. Haenen1, G.H. Koek2 ,1, A. Bast1 et al 1 Maastricht University, The Netherlands, 2Maastricht University Medical Centre, The Netherlands Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome which is caused by the unhealthy Western lifestyle. It includes a spectrum of disorders ranging from steatosis to steatohepatitis, cirrhosis and hepatocellular carcinoma. Twenty to thirty percent of patients with steatosis develop non-alcoholic steatohepatitis (NASH). In the development of NASH oxidative stress plays a crucial role. NASH can develop without other features of the metabolic syndrome in patients with citrin deficiency. In
S160
Altered redox status and signalling in dermal fibroblasts from psoriatic patients V.V. Barygina*1, T. Lotti2, M. Becatti1, C. Fiorillo1, N. Taddei1 1 University of Florence, Italy, 2University "G.Marconi", Italy Psoriasis is an inflammatory skin disease, characterized by hyper-proliferation and incomplete differentiation of keratinocytes accompanied by inflammation of both epidermis and derma. In psoriasis, the involvement of oxidative stress has been found in blood and skin. However, few publications are currently available on the redox status of dermal fibroblasts in psoriatic patients. Present study included 6 patients with moderate psoriasis and 5 healthy controls matched for sex and age. From each psoriatic patients the primary cultures of
We found recently that hydroxyl radical (•OH) could be produced by chlorinated quinones (CBQs) and hydrogen peroxide (H2O2) via a novel metal-independent nucleophilic substitution and homolytical decomposition mechanism. However, it is still not clear how much •OH can be produced by this unusual •OH generating system. In this work, the •OH yield by CBQs and H2O2 was quantitatively determined using salicylic acid (SA) hydroxylation method coupled with HPLCelectrochemical detection, and compared with that produced by the classic iron-mediated Fenton system (Fe(II)EDTA/H2O2). We found that •OH was produced both time- and dose-dependently on CBQs, H2O2 and SA as well as pH. Interestingly, the •OH production by CBQs/H2O2 was found to be maximal under weakly alkaline condition (pH, ≈ 8.0) while that by the classic Fenton system was maximal under weakly acidic condition (pH, ≈ 6.0) The maximal •OH yield by CBQs/H2O2 was found to be dependent on both the number and the position of the chlorine substitution. Surprisingly, some metal-independent •OH producing CBQs/H2O2 system was found to generate even more • OH than the classic Fenton system under the optimal conditions. In conclusion, CBQ/H2O2 system is a very efficient •OH producing system, and we suggest that they can be used to substitute the classic Fenton system to produce •OH for various biological studies, especially when there is a need to exclude the presence of transition metal ions. Keywords: Hydroxyl radical, chlorinated quinones, salicylic acid hydroxylation, 2,3-/2,5-dihydroxybenzoic acid doi:10.1016/j.freeradbiomed.2012.08.334 [0573] Physiological effects of the reactive sulphur species (RSS) allicin from garlic A.J. Slusarenko*, M.C.H. Gruhlke RWTH Aachen University, Germany Allicin (diallyl thiosulfinate) is a naturally occurring reactive sulphur species (RSS)1. Allicin is volatile, antimicrobial and is produced in garlic when the tissues are damaged; giving freshly cut garlic its characteristic odour. Allicin is readily membrane-permeable and a prooxidant which undergoes thiol-disulphide exchange reactions with free thiol groups in proteins. It is thought that these properties are the basis of its antimicrobial action. Allicin is part of the defence repertoire employed by garlic to defend itself against pathogens and pests. We have shown the effectiveness of pure synthetic allicin and garlic juice containing allicin, against a range 2 of plant pathogenic organisms in vitro and in planta . In
S159
addition to reacting with thiol groups in proteins we have shown that synthetic allicin induces apoptosis in yeast by altering the glutathione pool (GSH:GSSG) and disturbing the cell’s electrochemical potential3. We are currently characterizing the cellular details of the allicin-induced route into apoptosis in baker’s yeast (Saccharomyces cerevisiae) as a model fungus. Our results show that protein kinase A (PKA) activity is necessary for allicininduced apoptosis to occur and appears to coordinate apoptosis inducing factor (AIF) activity. Furthermore, we have shown that in a concentration-dependent manner, allicin permeabilizes cell membranes and is more active on a molar basis than dimethylsulfoxide (DMSO) in this respect. Thus, allicin not only has potential for development in plant protection, but it is also a useful RSS tool to investigate fungal apoptosis and various aspects of cell physiology. 1
Gruhlke and Slusarenko (2012) Plant Physiology and Biochemistry. http://dx.doi.org/10.1016/j.plaphy.2012.03.016 2 Curtis, et al. (2004) Physiological and Molecular Plant Pathology 65, 79-89 3 Gruhlke et al. (2010) Free Radical Biology and Medicine 49, 1916-1924, doi:10.1016/j.freeradbiomed.2010.09.019 Keywords: glutathione, species, allicin
apoptosis,
reactive
citrin deficiency there is a defect in the mitochondrial aspartate-glutamate carrier, leading to compensatory up regulation of the malate-citrate carrier, which causes an increase in the cytosolic citrate concentration. Interestingly, citrate is a metabolic intermediate that itself cannot be oxidized by the cell. We hypothesized that increased citrate levels, caused by an abundance of fatty acids, contribute to the development of NASH by an increase of oxidative stress. Therefore the aim of this study was to investigate citrate levels in NASH patients and to investigate the effect of citrate on oxidative stress. In line with our hypothesis, we found significantly increased blood plasma levels of citrate in NASH patients. Moreover, addition of palmitic acid to the incubation medium of immortalized hepatocytes (HepG2 cells) was found to increase the intracellular citrate concentration. This was measured with the use of liquid chromatography-tandem mass spectrometry. Oxidative stress in HepG2 cells exposed to citrate was measured with the use of 2'7'- dichlorofluorescein (DCFH). Citrate was found to increase radical formation by the Fenton reaction, which is probably related to the ability of citrate to chelate iron. Based on these findings, we postulate that abundance of fatty acids in NASH results in elevated citrate levels. This can lead to an increase of oxidative stress which contributes to the development of NASH.
sulfur
doi:10.1016/j.freeradbiomed.2012.08.335
Keywords: Non-alcoholic Oxidative stress, Fatty acids
steatohepatitis,
Citrate,
doi:10.1016/j.freeradbiomed.2012.08.336 [0585] [0588] Increase of oxidative stress in NASH by increased levels of citrate B. van de Wier*1, G.R.M.M. Haenen1, G.H. Koek2 ,1, A. Bast1 et al 1 Maastricht University, The Netherlands, 2Maastricht University Medical Centre, The Netherlands Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome which is caused by the unhealthy Western lifestyle. It includes a spectrum of disorders ranging from steatosis to steatohepatitis, cirrhosis and hepatocellular carcinoma. Twenty to thirty percent of patients with steatosis develop non-alcoholic steatohepatitis (NASH). In the development of NASH oxidative stress plays a crucial role. NASH can develop without other features of the metabolic syndrome in patients with citrin deficiency. In
S160
Altered redox status and signalling in dermal fibroblasts from psoriatic patients V.V. Barygina*1, T. Lotti2, M. Becatti1, C. Fiorillo1, N. Taddei1 1 University of Florence, Italy, 2University "G.Marconi", Italy Psoriasis is an inflammatory skin disease, characterized by hyper-proliferation and incomplete differentiation of keratinocytes accompanied by inflammation of both epidermis and derma. In psoriasis, the involvement of oxidative stress has been found in blood and skin. However, few publications are currently available on the redox status of dermal fibroblasts in psoriatic patients. Present study included 6 patients with moderate psoriasis and 5 healthy controls matched for sex and age. From each psoriatic patients the primary cultures of