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B-carotene oxidation products inactivate protein kinase C: role of apocarotenals in oxidative stress
L!!!Ll
I 63
7,8 DIHYDRONJ~OFTK~RTNCAN PROTECT CELLS FROM FREE RADICAL MEDIATED DAMAGE
&even Gieseg, Tomas Cruz, Dylan Glubb, Ghassan Muzghal and Jacqueline Whybrow Department of Zoology, University of Canterbury, Christchurch, New Zealand. Neopterin and the reduced fan, 7,8 dihydroneopterln (78NP) are pteridies released from macrophages and monocytes when stimulated with y-interferon in-ho. The role of 78NP in intlammatory response is unknown though there is an enormous amount of information available on the use of these compounds as clinical markers of monocyte/macrophage activation. Our previous studies on low density lipoprotein oxidation showed 78Mp to be a potent antioxidant. The research reported here ezamined whether 78Np could protect cellular systems fhxn radical induced damage. Red blood cells or U937 cells (a monocyte like human cell line) were exposed to rxdicals generated by the presence of Fe“, hydrogen peroxide, hypocblotite or AAPH. The level of damage to the cells was significantly reduced by the presence of 78NP in the incubation media, as measured by cell lysis, lipid oxidation, and metabolic ilmction assays. These findings suggest the role of 78Np may be to protect macrophage cells from free radical damage in inflammatory sites in viv0
B-CAROTENE OXIDATION PRODUCTS INACTIVATE PROTEIN KINASE c: ROLE OF APOCAR0TENAL.S IN
OXIDATIVE STRESS R.sTp” Gwui+J.
S. Bertram ad y. I. ~~~,S~C&ckofh4td., ; Cmm Ra 12, Hmdulu; Tuf3.sUnrv. Sch. of
Initially, we observed that certain preparations of beta-carotene (BC) inactivated both kinase activity and phorbol ester binding of PKC (IC, = 0.5 to 5 uM). However, a detailed study revealed that this effect was not caused by the. parent compound, but by its oxidation roducts formed during storage. These polar products were aLso sormed from BC by
autooxldation ooxrrln g spontaneously in an oxygen atmosphere at 60°C or Initiated by azo-bls-isobutyronitrlle. These products modified four cyateine residues in PKC. The BC oxidation products inserted into Ii somes still promoted the inactivation of membrane-bound PK r . The membrane-insert@ zinc-gngers present within the PKC regulatory domain provided specificity for this modification. BC autooxidation in liposomes made of peroxidiible lipids resulted in a slow rate of inactivation of PKC. Vitamin E inhibited this process, while it failed to block the effea of preformed oxidation products. The BC oxidation products, E&epoxy-BC and b-apo-lZkarotena1, were found to be ineffective. On the other hand, b-apo-15-carotenal (retinal) inactivated PKC ffC& = 1 to 5 uM). However, retinol and retinoic acid did not induce this inactivation. Borohydride reduction of a mixture of oxidation products or retinal lost their ability to inactivate PKC. Therefore, it is possible that BC oxidation products such as apocarotenals might exert antitumor promoting activity through an inactivation of PKC.
I 65 I HYPOXIA AND OXIDATIVE DEFENSES IN PLASMA OF THE SEA BASS,DICENTRARCHUS LABRAX -
hkina
II Unru , Nap&
Pmlllcci’ od G&am Ciprcia’. (1) r-mail-cisrcia @aunin4.it.
1. Depi zidogy,
It has been proposed that the oxidative damage reflects an imbalance between the production of oxidants and removal or scavenging of those oxidants. Specially adapted ensymes such as superoxide dismutases, catalase and glutathione peroxidams prevent the formation of oxyradicals. Non-enzymatic defenses in&de the alpha-tocopherol, beta-carotene, ascorbic acid and glutathione. In order to evaluate the non-ensymatic and enzymatic interaction after stress induction by hypoxia-reoxygenation, we studied alpha-tocopherol, ascorbic acid and glutathione peroxidase in the bred sea bass, &Dicentrarchus labrax, of different size in both exposed and not exposed to hypoxia for 40 minutes at 1 mg/l of oxygen and recovered at standard condition (8 n&l). 5ea bass exposed to normoxia after hypoxia were maintained at reoxygenation for 1530 and 60 minutes. Plasmatic alpha-tocopherol and ascorbic acid were analyzed by high performance liquid chromatography;plasmatic glutathione pemxidase by enzymatic assay. Results showed a linear correlation between ghrtathione peroxidase and size of the sea bass. The plasmatic alpha-tocopherol and ascorbic acid content in the sea bass exposed to hypoxia-reoxygenation (40 min-15 min) were lower than sea bass maintained at nonnoxta and were inversely correlated to the size and glutathione peroxidase. No difference are releaved at 30 and 60 minutes of reoxygenation. The results indicate that the glutathione peroxidare is directly coupled to alpha-tocopherol recycling by its ability to regenerate ascorbic acid and suggest the reduction of the assayed plasmatic alpha-tocopheml and ascorbic acid might be consequence and parameter of its utilization throughout the oxyradicals process.
s32
OXYGEN
MODULATION OF VASCULAR EXTRACELLULAR SOD EXPRESSION BY ANGIOTENSIN II AND HYPERTENSION Fukai, Martin R. Siegfrkd, Mado U&o-F&i, .j%$ FGm;Thru A major form of vascular superoxide dismutase is the extracellular SOD k&OD). Because angiotensin II (AU and hypertension increase vascular oxidant stress, we examined how these might influence ecSOD expression. In mice, infusion of AII (l.lmg/kg/day X 7 days) increased systolic blood pressure from 107L3to 15233 nunHg and increased aortic ecSDDexpression by J-fold as assessed by Western analysis. BNAse protection assays showed that AI1 infusion also increased ecSOD mBNA expression by 3-fold. Induction of ecSDD during AE infusion was not due to hypertension alone, as norepinephrine infusion (5.6 m&kg/day), which increased SBP to 14%16 nunFIg, did not affect ecSOD expression. Similarly, exposure of mouse aortas to AII (100 nM) in organoid culture doubled ecSOD expression. In organoid culture, AII induced upregulation of ecSOD was prevented by Losartan fluM) and the specific p42/44 MEK inhibitor I’D985059 (3OuM). AI1 activates the NADH/NADPH oxidase however, diphenylene iodonium (DPI, 10&i), an inhibitor of this oxidase, did not prevent ecSOD induction by AIL Further, DPI did not inhibit phosphorylation of p42/44MAF kinase caused by AII, suggesting that AII activation of pQ2/44MAFK pathway is NADH/NADFH oxidase-independent. Finally, we found that AI1 moderately increased transcriptional rate (as assessed by nuclear run-on analysis) and markedly increased ecSOD mBNA stability in cultured human aortic smooth muscle cells. Thus, these studies show that AII increases ecSOD expression independent of hypertension, and that this involves both an increase in ecSOD transcription and stabilization of ecSOD rnBNA. This effect of AB on ec?OD expression may modulate vascular oxidative state in pathological processes in which the renin-angiotensin system is activated.
’ 9
8
I 63
7,8 DIHYDRONJ~OFTK~RTNCAN PROTECT CELLS FROM FREE RADICAL MEDIATED DAMAGE
&even Gieseg, Tomas Cruz, Dylan Glubb, Ghassan Muzghal and Jacqueline Whybrow Department of Zoology, University of Canterbury, Christchurch, New Zealand. Neopterin and the reduced fan, 7,8 dihydroneopterln (78NP) are pteridies released from macrophages and monocytes when stimulated with y-interferon in-ho. The role of 78NP in intlammatory response is unknown though there is an enormous amount of information available on the use of these compounds as clinical markers of monocyte/macrophage activation. Our previous studies on low density lipoprotein oxidation showed 78Mp to be a potent antioxidant. The research reported here ezamined whether 78Np could protect cellular systems fhxn radical induced damage. Red blood cells or U937 cells (a monocyte like human cell line) were exposed to rxdicals generated by the presence of Fe“, hydrogen peroxide, hypocblotite or AAPH. The level of damage to the cells was significantly reduced by the presence of 78NP in the incubation media, as measured by cell lysis, lipid oxidation, and metabolic ilmction assays. These findings suggest the role of 78Np may be to protect macrophage cells from free radical damage in inflammatory sites in viv0
B-CAROTENE OXIDATION PRODUCTS INACTIVATE PROTEIN KINASE c: ROLE OF APOCAR0TENAL.S IN
OXIDATIVE STRESS R.sTp” Gwui+J.
S. Bertram ad y. I. ~~~,S~C&ckofh4td., ; Cmm Ra 12, Hmdulu; Tuf3.sUnrv. Sch. of
Initially, we observed that certain preparations of beta-carotene (BC) inactivated both kinase activity and phorbol ester binding of PKC (IC, = 0.5 to 5 uM). However, a detailed study revealed that this effect was not caused by the. parent compound, but by its oxidation roducts formed during storage. These polar products were aLso sormed from BC by
autooxldation ooxrrln g spontaneously in an oxygen atmosphere at 60°C or Initiated by azo-bls-isobutyronitrlle. These products modified four cyateine residues in PKC. The BC oxidation products inserted into Ii somes still promoted the inactivation of membrane-bound PK r . The membrane-insert@ zinc-gngers present within the PKC regulatory domain provided specificity for this modification. BC autooxidation in liposomes made of peroxidiible lipids resulted in a slow rate of inactivation of PKC. Vitamin E inhibited this process, while it failed to block the effea of preformed oxidation products. The BC oxidation products, E&epoxy-BC and b-apo-lZkarotena1, were found to be ineffective. On the other hand, b-apo-15-carotenal (retinal) inactivated PKC ffC& = 1 to 5 uM). However, retinol and retinoic acid did not induce this inactivation. Borohydride reduction of a mixture of oxidation products or retinal lost their ability to inactivate PKC. Therefore, it is possible that BC oxidation products such as apocarotenals might exert antitumor promoting activity through an inactivation of PKC.
I 65 I HYPOXIA AND OXIDATIVE DEFENSES IN PLASMA OF THE SEA BASS,DICENTRARCHUS LABRAX -
hkina
II Unru , Nap&
Pmlllcci’ od G&am Ciprcia’. (1) r-mail-cisrcia @aunin4.it.
1. Depi zidogy,
It has been proposed that the oxidative damage reflects an imbalance between the production of oxidants and removal or scavenging of those oxidants. Specially adapted ensymes such as superoxide dismutases, catalase and glutathione peroxidams prevent the formation of oxyradicals. Non-enzymatic defenses in&de the alpha-tocopherol, beta-carotene, ascorbic acid and glutathione. In order to evaluate the non-ensymatic and enzymatic interaction after stress induction by hypoxia-reoxygenation, we studied alpha-tocopherol, ascorbic acid and glutathione peroxidase in the bred sea bass, &Dicentrarchus labrax, of different size in both exposed and not exposed to hypoxia for 40 minutes at 1 mg/l of oxygen and recovered at standard condition (8 n&l). 5ea bass exposed to normoxia after hypoxia were maintained at reoxygenation for 1530 and 60 minutes. Plasmatic alpha-tocopherol and ascorbic acid were analyzed by high performance liquid chromatography;plasmatic glutathione pemxidase by enzymatic assay. Results showed a linear correlation between ghrtathione peroxidase and size of the sea bass. The plasmatic alpha-tocopherol and ascorbic acid content in the sea bass exposed to hypoxia-reoxygenation (40 min-15 min) were lower than sea bass maintained at nonnoxta and were inversely correlated to the size and glutathione peroxidase. No difference are releaved at 30 and 60 minutes of reoxygenation. The results indicate that the glutathione peroxidare is directly coupled to alpha-tocopherol recycling by its ability to regenerate ascorbic acid and suggest the reduction of the assayed plasmatic alpha-tocopheml and ascorbic acid might be consequence and parameter of its utilization throughout the oxyradicals process.
s32
OXYGEN
MODULATION OF VASCULAR EXTRACELLULAR SOD EXPRESSION BY ANGIOTENSIN II AND HYPERTENSION Fukai, Martin R. Siegfrkd, Mado U&o-F&i, .j%$ FGm;Thru A major form of vascular superoxide dismutase is the extracellular SOD k&OD). Because angiotensin II (AU and hypertension increase vascular oxidant stress, we examined how these might influence ecSOD expression. In mice, infusion of AII (l.lmg/kg/day X 7 days) increased systolic blood pressure from 107L3to 15233 nunHg and increased aortic ecSDDexpression by J-fold as assessed by Western analysis. BNAse protection assays showed that AI1 infusion also increased ecSOD mBNA expression by 3-fold. Induction of ecSDD during AE infusion was not due to hypertension alone, as norepinephrine infusion (5.6 m&kg/day), which increased SBP to 14%16 nunFIg, did not affect ecSOD expression. Similarly, exposure of mouse aortas to AII (100 nM) in organoid culture doubled ecSOD expression. In organoid culture, AII induced upregulation of ecSOD was prevented by Losartan fluM) and the specific p42/44 MEK inhibitor I’D985059 (3OuM). AI1 activates the NADH/NADPH oxidase however, diphenylene iodonium (DPI, 10&i), an inhibitor of this oxidase, did not prevent ecSOD induction by AIL Further, DPI did not inhibit phosphorylation of p42/44MAF kinase caused by AII, suggesting that AII activation of pQ2/44MAFK pathway is NADH/NADFH oxidase-independent. Finally, we found that AI1 moderately increased transcriptional rate (as assessed by nuclear run-on analysis) and markedly increased ecSOD mBNA stability in cultured human aortic smooth muscle cells. Thus, these studies show that AII increases ecSOD expression independent of hypertension, and that this involves both an increase in ecSOD transcription and stabilization of ecSOD rnBNA. This effect of AB on ec?OD expression may modulate vascular oxidative state in pathological processes in which the renin-angiotensin system is activated.
’ 9
8