- Email: [email protected]
SPC promoter-driven expression of HO-1 exacerbates hyperoxic lung injury in vivo
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ANTIRADICAL ACTIVITY OF DIFFERENT THIOLIC COMPOUNDS EVALUATED BY EPR Andrea Bnrbiwi, Marilena Lais, Ad&do
-Chrcol .Dept. D Stolofl
-
Dept. of Chemis:~, Untisily
Ciao%, MiEoin
ofFernma,My.
Thiolic scavengers represent a relevant defense line against oxygen radical damage in biological systems. We evaluated and compared the Scavenging efficiency of different thiolic compounds by electron paramagnetic resonance (EPR). The comPounds tested were ambmxol (4-(Z-amino- 3,s dibromophenyl)- methyl amino cyclohexanol) (Zamino- 3, Sdibromobenzyl amine), N-acetyl L-cysteine (NAC), S-carboxymethyl cysteine lysine salt (SCMC), reduced glutathione (GSH) and natural sulphureous water (“Breta”)from a spring near Rio10 Terme, Ravenna (Italy). Radicals were generated by the Fenton reaction, obtained by Mohr’s salt (ammonium ferrous sulphate) and hydrogen peroxide. EPR analysis were performed in a flat cell placed in the TM 110 cavity of a Bruker ER200 spectrometer, at room temperature, using the spin-trapping technique with 5,5- dimethylI-pyrroline N-oxide (DMPO). All compounds, except ambroxol, exhibit different degrees of HO-scavenging ability. Analysis of EPR spectral patterns reveals that the HO-scavenging kinetics of NAC, SCMC and GSH are competitive in comparison to DMPO, namely they are all faster to capture hydroxyl radicals. The EPR patterns in presence of ambroxol still exhibits DMPO-hydroxyl adducts, therefore the HO-capturing ability of ambroxol matches that of DMPO, making it an unefficient scavenger in comparison to the others. It is nevertheless rather difficult to establish a graded comparison of scavenging efficiency among the other compounds. Natural sulphureous spring water “Breta”abnormally accelerated the trapping kinetics of HO by DMFO. The results are discussed in relation to possible synergic use of thiolic scavengers in therapy of lung diseases.
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Vegetal Cu/Zn-Superoxide Dismutase Protects Against HIV-lInduced Pro-Intlammatory Reactions and Limit HIV-1 Replication and Spreading Eric Postaire*, Bernard Dugas, Alphonse Calenda, Virginie Sivan, ElianLati,Michel Brack, and Jacques Sauzibres. FRACTALES Biotech S.A., CEA-INRA, Laboratoire Mixte de Radiobiologie AppliquCe, 78352 Jouy en Josas, CIRDC, 92350 Le Plessis Robinson, France The influence of increased oxidative stress during viral infections has gained momentum given the accumulation of evidence regarding the role of ROS and NO in the pathogenesis of infection with the human immunodeficiency virus (HIV). A particular attention will be focussed on HIV-l-infection The following evidences are discussed: (1) the effect of the virus on the activation of phagocytic cells to release ROS, NO, proinflammatory cytokines (e.g. TNF) and/or chemokines (RANTES, MIP-la); (2) the effect of the virus on the pro/antioxidant balance in host cells, with a special attention to virally induced inhibition of anti-oxidant enzymes; (3) the effects of the redox state of the cells on the immunological status of lymphoid cells (cytokine production, apoptosis etc.) and on the genetic composition of the virus as well as viral replication and spreading; and (4) efficacy of anti-oxidants as therapeutic agent in viral diseases. Recent experiments clearly indicated that Cu/Zn-SOD inhibited the cellular immuno-redox alteration induced HIV-I-Tat and envelope proteins (gp160, gp120 and gp41) and limited the replication of the virus in infected cells. In addition to this pharmacological effect this CuiZn-SOD not only strengthened the anti-viral effect of the conventional antiretroviral therapy (AZT, dd1, Ritonavir, Saquinavir etc.), but also reduced the toxicity of these products (especially AZT) without affecting the anti-viral properties of the product. Using, a new bio-vector we demonstrate this CWZn-SOD can be delivered orally and could be used in clinic especially in the treatment of viral infectious diseases and/or in the detoxification of drugs.
El55 SPC PROMOTER-DRIVEN EXPRESSION OF HO-l EXACERBATES HYPEROXIC LUNG INJURY IN WV0 ~~~~~s~~~~~~h~~~~~l~~,s~~~~~~~ Heme oxygenace (HO) catalyzes the first step in metabolism of heme to biliverdin and biiirubin, with concomitant production of CO and liberation of heme-bound iron. Some investigators have proposed that the inducible &form, HO-l, may contribute to adaptive responses to oxidant stresses, in part through the production of bilirubin, which can exhibit antioxidant activities. To test this hypothesis in vivo, we generated a line of transgenic mice that express the rat spleen HO-1 cDNA driven by a human surfactant protein C promoter. HO activities 660+/-71 vs. 181+/-15 pmol/min/g tissue) and HO-l protein and mRNA levels were greater in the lungs of the transgenic animals than in lungs from control animals The transgenic animals were more susceptible to lung injury in response to exposure to >95%02 as w by lung weights (12.17+/-2.&l vs 7.76+/X1.78mg lung/ g animal by 96 h). Hyperoxia increased lung HO activities and HO-1 mRNA levels in the control animals, as has been reported previously, but message levels and HO activities were diminished by hyperoxia in the lungs of transgenic animals (242+/-82 pmol/min/g after 48 h). HO1 expression in air-breathing tran~enic animals appeared by immunohistochemistry to be predominantly in lung type II cells, but expression in type Jl cells was largely lost by 48 h or more of hypemxia. Increased HO-1 protein in both bansgenic and wild-type animals exposed to hyperoxia appeared to be associated almost exclusively with macrophages. The present data indicate that although increased expression of HO-1 may contribute to antioddant defense function, cell specificity and regulation of expression in coordination with other stress response genes are likely to be important factors. Supported by HDOl124 and HD27823.
OXYGEN
ANTIOXIDANT ACI’IVITY OF RESVERATROL ANALOGS IN PHOSPHOLIPID OXIDATION and Cherk M. Milnar, Department ofChemists & w retry, Duquesne Uniwsi?y, Pitt&u-h PA
Resveratrol (trans_3,4’5_trihydroxystilbene),a naturally occurringphenolic compound found in grape skin and wines, has been shown to have potent activity as a membmne antioxidant. We have examined a series of mono- and dihydroxy stilbene analogs of resveratrol in an attempt to defme the structural determinants of this natural prcduct which contribute to its antioxidant efficacy. Both truns4hydroxy and fruns-4,4’-dihydroxystillxmederivatives show significant inhibition of li id vesicle oxidation initiated by a water-soluble azoinitiator. A)t a concentration of 0.01 mM, resveratro~ 4hydroxy-, and 4,4’dihydroxystilbene gave inhibition petiods of 55,135 and 100 minutes, respectively. Data will be also be presented comparing the effects of these antioxidants using lipid-soluble initiators. Our results demonstrate that molecular simplifkations of resveratrol possess equipotent antioxidant activity in a lipid membrane system.
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ANTIRADICAL ACTIVITY OF DIFFERENT THIOLIC COMPOUNDS EVALUATED BY EPR Andrea Bnrbiwi, Marilena Lais, Ad&do
-Chrcol .Dept. D Stolofl
-
Dept. of Chemis:~, Untisily
Ciao%, MiEoin
ofFernma,My.
Thiolic scavengers represent a relevant defense line against oxygen radical damage in biological systems. We evaluated and compared the Scavenging efficiency of different thiolic compounds by electron paramagnetic resonance (EPR). The comPounds tested were ambmxol (4-(Z-amino- 3,s dibromophenyl)- methyl amino cyclohexanol) (Zamino- 3, Sdibromobenzyl amine), N-acetyl L-cysteine (NAC), S-carboxymethyl cysteine lysine salt (SCMC), reduced glutathione (GSH) and natural sulphureous water (“Breta”)from a spring near Rio10 Terme, Ravenna (Italy). Radicals were generated by the Fenton reaction, obtained by Mohr’s salt (ammonium ferrous sulphate) and hydrogen peroxide. EPR analysis were performed in a flat cell placed in the TM 110 cavity of a Bruker ER200 spectrometer, at room temperature, using the spin-trapping technique with 5,5- dimethylI-pyrroline N-oxide (DMPO). All compounds, except ambroxol, exhibit different degrees of HO-scavenging ability. Analysis of EPR spectral patterns reveals that the HO-scavenging kinetics of NAC, SCMC and GSH are competitive in comparison to DMPO, namely they are all faster to capture hydroxyl radicals. The EPR patterns in presence of ambroxol still exhibits DMPO-hydroxyl adducts, therefore the HO-capturing ability of ambroxol matches that of DMPO, making it an unefficient scavenger in comparison to the others. It is nevertheless rather difficult to establish a graded comparison of scavenging efficiency among the other compounds. Natural sulphureous spring water “Breta”abnormally accelerated the trapping kinetics of HO by DMFO. The results are discussed in relation to possible synergic use of thiolic scavengers in therapy of lung diseases.
I
Vegetal Cu/Zn-Superoxide Dismutase Protects Against HIV-lInduced Pro-Intlammatory Reactions and Limit HIV-1 Replication and Spreading Eric Postaire*, Bernard Dugas, Alphonse Calenda, Virginie Sivan, ElianLati,Michel Brack, and Jacques Sauzibres. FRACTALES Biotech S.A., CEA-INRA, Laboratoire Mixte de Radiobiologie AppliquCe, 78352 Jouy en Josas, CIRDC, 92350 Le Plessis Robinson, France The influence of increased oxidative stress during viral infections has gained momentum given the accumulation of evidence regarding the role of ROS and NO in the pathogenesis of infection with the human immunodeficiency virus (HIV). A particular attention will be focussed on HIV-l-infection The following evidences are discussed: (1) the effect of the virus on the activation of phagocytic cells to release ROS, NO, proinflammatory cytokines (e.g. TNF) and/or chemokines (RANTES, MIP-la); (2) the effect of the virus on the pro/antioxidant balance in host cells, with a special attention to virally induced inhibition of anti-oxidant enzymes; (3) the effects of the redox state of the cells on the immunological status of lymphoid cells (cytokine production, apoptosis etc.) and on the genetic composition of the virus as well as viral replication and spreading; and (4) efficacy of anti-oxidants as therapeutic agent in viral diseases. Recent experiments clearly indicated that Cu/Zn-SOD inhibited the cellular immuno-redox alteration induced HIV-I-Tat and envelope proteins (gp160, gp120 and gp41) and limited the replication of the virus in infected cells. In addition to this pharmacological effect this CuiZn-SOD not only strengthened the anti-viral effect of the conventional antiretroviral therapy (AZT, dd1, Ritonavir, Saquinavir etc.), but also reduced the toxicity of these products (especially AZT) without affecting the anti-viral properties of the product. Using, a new bio-vector we demonstrate this CWZn-SOD can be delivered orally and could be used in clinic especially in the treatment of viral infectious diseases and/or in the detoxification of drugs.
El55 SPC PROMOTER-DRIVEN EXPRESSION OF HO-l EXACERBATES HYPEROXIC LUNG INJURY IN WV0 ~~~~~s~~~~~~h~~~~~l~~,s~~~~~~~ Heme oxygenace (HO) catalyzes the first step in metabolism of heme to biliverdin and biiirubin, with concomitant production of CO and liberation of heme-bound iron. Some investigators have proposed that the inducible &form, HO-l, may contribute to adaptive responses to oxidant stresses, in part through the production of bilirubin, which can exhibit antioxidant activities. To test this hypothesis in vivo, we generated a line of transgenic mice that express the rat spleen HO-1 cDNA driven by a human surfactant protein C promoter. HO activities 660+/-71 vs. 181+/-15 pmol/min/g tissue) and HO-l protein and mRNA levels were greater in the lungs of the transgenic animals than in lungs from control animals The transgenic animals were more susceptible to lung injury in response to exposure to >95%02 as w by lung weights (12.17+/-2.&l vs 7.76+/X1.78mg lung/ g animal by 96 h). Hyperoxia increased lung HO activities and HO-1 mRNA levels in the control animals, as has been reported previously, but message levels and HO activities were diminished by hyperoxia in the lungs of transgenic animals (242+/-82 pmol/min/g after 48 h). HO1 expression in air-breathing tran~enic animals appeared by immunohistochemistry to be predominantly in lung type II cells, but expression in type Jl cells was largely lost by 48 h or more of hypemxia. Increased HO-1 protein in both bansgenic and wild-type animals exposed to hyperoxia appeared to be associated almost exclusively with macrophages. The present data indicate that although increased expression of HO-1 may contribute to antioddant defense function, cell specificity and regulation of expression in coordination with other stress response genes are likely to be important factors. Supported by HDOl124 and HD27823.
OXYGEN
ANTIOXIDANT ACI’IVITY OF RESVERATROL ANALOGS IN PHOSPHOLIPID OXIDATION and Cherk M. Milnar, Department ofChemists & w retry, Duquesne Uniwsi?y, Pitt&u-h PA
Resveratrol (trans_3,4’5_trihydroxystilbene),a naturally occurringphenolic compound found in grape skin and wines, has been shown to have potent activity as a membmne antioxidant. We have examined a series of mono- and dihydroxy stilbene analogs of resveratrol in an attempt to defme the structural determinants of this natural prcduct which contribute to its antioxidant efficacy. Both truns4hydroxy and fruns-4,4’-dihydroxystillxmederivatives show significant inhibition of li id vesicle oxidation initiated by a water-soluble azoinitiator. A)t a concentration of 0.01 mM, resveratro~ 4hydroxy-, and 4,4’dihydroxystilbene gave inhibition petiods of 55,135 and 100 minutes, respectively. Data will be also be presented comparing the effects of these antioxidants using lipid-soluble initiators. Our results demonstrate that molecular simplifkations of resveratrol possess equipotent antioxidant activity in a lipid membrane system.
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