- Email: [email protected]
Enrichment of endothelial cells with carotenoid does not alter LDL oxidation or peroxide sensitivity
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ENRICHMENT OF ENDOTHELIAL CELLS WITH CAROTENOID DOES NOT ALTER LDL OXIDATION PEROXIDE SENSITIVITY D W Morel and E.H. Harrison.. S&&s in Phila., Philadelphia, USDA, Beltsvilte, MD
Our previous studies demonstrated that LDL enriched with b-carotene, but not with lutein or lycopene, was protected from endothelial cell-mediated oxidation (FRBM26:1239, 1999). We now investigated the possibility that these carotenoids could act as celular antioxidants and inhibit cell-mediated LDL oxidation or protect cells from hydrogen peroxide mediated toxicity. Cell enrichment with specific carotenoids was achieved by culturing them in the presence of medium containing 10% FBS pre-enriched by addition of carotenoid in solvent (THF for b-carotene and lycopene, ethanol for lutein). A time (with maximum after 2 days) and concentration-dependent accumulation of carotenoid in cells reaching 100-400 ng carotenoid/mg cell protein was seen. Cells were incubated with human LDL, with samples analyzed at various times for lipid hydroperoxides by ferrous ion-xylenol orange reactivity. Enrichment of cells with b-carotene or lutein did not alter the time course of LDL oxidation mediated by these cells. In other experiments, cells were enriched with carotenoid, labelled with radioactive adenine, then exposed to increasing concentrations of hydrogen peroxide. Toxicity, measured as release of adenine-derived radiolabel into medium (2-4 h), increased markedly between 0.75-2 mM hydrogen peroxide and was unaltered by cell enrichment with carotenoid. These results suggest that increases in individual carotenoid concentrations, at least at the levels achieved here, do not protect endothelial cells from oxidative stress or alter their ability to oxidize LDL.
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STATUS IN INFLUENZA
KSA Mossanda,EB -___-_______
INFECTED PATIENTS
Dabula,E Mokgawa and HF Joubert
Chemical Pathology Dept.PO Box 227 MEDUNSA-South
Afric
Background: -___ ____I Various strains of Influenza are a major cause of illness and clinical complications of pneumonia,chronic heart and lung disease in the world. If these clinical features are well defined, a little however is known about biochemical aspects and treatment which is more prophylactic with a trivalent influenza vaccine and vit C and E used as antioxidant. Toxic reactive oxygen metabolites released from phagocytic leucocytes during influenza activity may overwhelm the protective enzymic and non-enzymic antioxidant. C&j~tives: To evaluate whether antioxidant status is affected by the influenza infection and justify the effective&s of an antioxidant treatment. _ Materials & Methods: Antioxidant status was performed 6y_evaftiafitig-I&%-biomarkers:Superoxide dismutase (SOD),Glutathione peroxidase(GPx),Malondialdhyde(MDA) and Total antioxidant capacity(TAC) using Randox kits commercially available and vit E and A by HPLC in 20 patients admitted for Influenza infection. Results: Low activity was observed for SOD and GPx in -____-_ contrast with normal values of TAC, vit E and A. SOD and GPx were higher in males than in females. Conclusion: -__ ______ Using F test for the significance of multipre correlation coefficient between these biomarkers considered as predictor variables,we will know whether or not they contribute separately or together to the effectiveness of antioxidant orevention.
1 IN UVA LIGHT
Ufe Obermueller-TeviG Pal Istuan Francz, \ut’r en Frank, Hans Konrad University of Hohenheim, Stutt,qnrt D-70593 s ERMANY
Biesafski.
&Carotene has often been discussed as a means to reduce the risk of oxidative damage in sunlight exposed skin. Several in vim studies show beneficial effects of fl-carotene but there are only few data about its mechanism of action in vitro. We studied the antioxidative potential of &carotene in UVA light irradiated human skin fibroblasts. Surprisingly, we found a strong pro-oxidative effect of fl-carotene. HFP-1 fibroblasts were preincubated with fl-carotene (0.5 or 5 PM), or vehicle alone. For solubilization methyl-fl-cyclodextrin was used. Cells were irradiated with sub-erythemal WA light (20 J/cd), or were sham irradiated. Using the induction of haem oxygenase-1 (HO-l) as an accepted marker for oxidative stress, we found a strong enhancement of WA activated HO-1 expression by fl-carotene on both protein (Western blot) and mRNA (Northern blot) levels. The fl-carotene enhanced HO-1 induction was clearly suppressed by concomitant addition of vitamin E (10 or 25 FM) but only moderately by vitamin C (50 or 100 KM). From our results we conclude that fl-carotene has pro-oxidative properties in membranes of human skin fibroblasts exposed to WA light. We assume that fl-carotene or any photo-degradation products of fl-carotene may act as photosensitizers.
s40
I
ANTIOXIDANT OR
Dept. Pharmaceutical Sciences, Univ. ofthe PA, and Human Nutrition Resenrch Center,
PRO-OXIDATIVE EFFECT OF &CAROTENE EXPOSED HUMAN SKIN FIBROBLASTS
93
OXYGEN
CYTOSOLIC NADP+-SPECIFIC ISOCITRATE DEHYDROGENASE AS AN ANTIOXIDANT ENZYME! Jeen-Woo Park, Su Min Lee and Tae Lin Huh Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea The cytosolic form of NADP’-specific isocitrate dehydrogenase @PC) in mammalian cells produces NADPH, which is an essential reducing equivalent for the antioxidant system. To determine the protective role of IDPc against cytotoxicity induced by oxidative stress, stable transfectants of NIH3T3 cells enriched in and partially deprived of IDPc were constructed with the mouse IDPc gene with the sense and antisense orientations, in which IDPc activities were 3-4 fold higher and 35% lower than in the parental cells carrying the vector alone, respectively. Upon exposure to oxidative stress, the viability was lower and lipid peroxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the transfectant overexpressing IDPc exhibites enhanced resistance against oxidative stress compared to control cells. Although the activities of major antioxidant enzymes were comparable in all cell-lines, the ratio of GSSG to total glutathione was significantly higher in the cell-line expressing the lower level of IDPc. This study provides direct evidence correlating the activities of IDPc and the maintenance of the cellular redox state, suggesting that IDPc plays an important role as an antioxidant enzyme.
’ 9
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92
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I
ENRICHMENT OF ENDOTHELIAL CELLS WITH CAROTENOID DOES NOT ALTER LDL OXIDATION PEROXIDE SENSITIVITY D W Morel and E.H. Harrison.. S&&s in Phila., Philadelphia, USDA, Beltsvilte, MD
Our previous studies demonstrated that LDL enriched with b-carotene, but not with lutein or lycopene, was protected from endothelial cell-mediated oxidation (FRBM26:1239, 1999). We now investigated the possibility that these carotenoids could act as celular antioxidants and inhibit cell-mediated LDL oxidation or protect cells from hydrogen peroxide mediated toxicity. Cell enrichment with specific carotenoids was achieved by culturing them in the presence of medium containing 10% FBS pre-enriched by addition of carotenoid in solvent (THF for b-carotene and lycopene, ethanol for lutein). A time (with maximum after 2 days) and concentration-dependent accumulation of carotenoid in cells reaching 100-400 ng carotenoid/mg cell protein was seen. Cells were incubated with human LDL, with samples analyzed at various times for lipid hydroperoxides by ferrous ion-xylenol orange reactivity. Enrichment of cells with b-carotene or lutein did not alter the time course of LDL oxidation mediated by these cells. In other experiments, cells were enriched with carotenoid, labelled with radioactive adenine, then exposed to increasing concentrations of hydrogen peroxide. Toxicity, measured as release of adenine-derived radiolabel into medium (2-4 h), increased markedly between 0.75-2 mM hydrogen peroxide and was unaltered by cell enrichment with carotenoid. These results suggest that increases in individual carotenoid concentrations, at least at the levels achieved here, do not protect endothelial cells from oxidative stress or alter their ability to oxidize LDL.
I 94 I
I
STATUS IN INFLUENZA
KSA Mossanda,EB -___-_______
INFECTED PATIENTS
Dabula,E Mokgawa and HF Joubert
Chemical Pathology Dept.PO Box 227 MEDUNSA-South
Afric
Background: -___ ____I Various strains of Influenza are a major cause of illness and clinical complications of pneumonia,chronic heart and lung disease in the world. If these clinical features are well defined, a little however is known about biochemical aspects and treatment which is more prophylactic with a trivalent influenza vaccine and vit C and E used as antioxidant. Toxic reactive oxygen metabolites released from phagocytic leucocytes during influenza activity may overwhelm the protective enzymic and non-enzymic antioxidant. C&j~tives: To evaluate whether antioxidant status is affected by the influenza infection and justify the effective&s of an antioxidant treatment. _ Materials & Methods: Antioxidant status was performed 6y_evaftiafitig-I&%-biomarkers:Superoxide dismutase (SOD),Glutathione peroxidase(GPx),Malondialdhyde(MDA) and Total antioxidant capacity(TAC) using Randox kits commercially available and vit E and A by HPLC in 20 patients admitted for Influenza infection. Results: Low activity was observed for SOD and GPx in -____-_ contrast with normal values of TAC, vit E and A. SOD and GPx were higher in males than in females. Conclusion: -__ ______ Using F test for the significance of multipre correlation coefficient between these biomarkers considered as predictor variables,we will know whether or not they contribute separately or together to the effectiveness of antioxidant orevention.
1 IN UVA LIGHT
Ufe Obermueller-TeviG Pal Istuan Francz, \ut’r en Frank, Hans Konrad University of Hohenheim, Stutt,qnrt D-70593 s ERMANY
Biesafski.
&Carotene has often been discussed as a means to reduce the risk of oxidative damage in sunlight exposed skin. Several in vim studies show beneficial effects of fl-carotene but there are only few data about its mechanism of action in vitro. We studied the antioxidative potential of &carotene in UVA light irradiated human skin fibroblasts. Surprisingly, we found a strong pro-oxidative effect of fl-carotene. HFP-1 fibroblasts were preincubated with fl-carotene (0.5 or 5 PM), or vehicle alone. For solubilization methyl-fl-cyclodextrin was used. Cells were irradiated with sub-erythemal WA light (20 J/cd), or were sham irradiated. Using the induction of haem oxygenase-1 (HO-l) as an accepted marker for oxidative stress, we found a strong enhancement of WA activated HO-1 expression by fl-carotene on both protein (Western blot) and mRNA (Northern blot) levels. The fl-carotene enhanced HO-1 induction was clearly suppressed by concomitant addition of vitamin E (10 or 25 FM) but only moderately by vitamin C (50 or 100 KM). From our results we conclude that fl-carotene has pro-oxidative properties in membranes of human skin fibroblasts exposed to WA light. We assume that fl-carotene or any photo-degradation products of fl-carotene may act as photosensitizers.
s40
I
ANTIOXIDANT OR
Dept. Pharmaceutical Sciences, Univ. ofthe PA, and Human Nutrition Resenrch Center,
PRO-OXIDATIVE EFFECT OF &CAROTENE EXPOSED HUMAN SKIN FIBROBLASTS
93
OXYGEN
CYTOSOLIC NADP+-SPECIFIC ISOCITRATE DEHYDROGENASE AS AN ANTIOXIDANT ENZYME! Jeen-Woo Park, Su Min Lee and Tae Lin Huh Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea The cytosolic form of NADP’-specific isocitrate dehydrogenase @PC) in mammalian cells produces NADPH, which is an essential reducing equivalent for the antioxidant system. To determine the protective role of IDPc against cytotoxicity induced by oxidative stress, stable transfectants of NIH3T3 cells enriched in and partially deprived of IDPc were constructed with the mouse IDPc gene with the sense and antisense orientations, in which IDPc activities were 3-4 fold higher and 35% lower than in the parental cells carrying the vector alone, respectively. Upon exposure to oxidative stress, the viability was lower and lipid peroxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the transfectant overexpressing IDPc exhibites enhanced resistance against oxidative stress compared to control cells. Although the activities of major antioxidant enzymes were comparable in all cell-lines, the ratio of GSSG to total glutathione was significantly higher in the cell-line expressing the lower level of IDPc. This study provides direct evidence correlating the activities of IDPc and the maintenance of the cellular redox state, suggesting that IDPc plays an important role as an antioxidant enzyme.
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