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Evaluation of antioxidant, prooxidant and genotoxic effects of caffeic acid: a in vivo study
EVALUATION
OF ANTIOXIDANT,
PROOXIDANT
AND
GENOTOXIC EFFECTS OF CAFFEIC ACID: A IN VIVO STUDY Ka I n Ckless. Michelle Susin, Ryane Hess, Cl.udm Simm, Rosa Ri&&-do-V& Fnrmncfuticns
Fnrmcin UNJSUL UFSC FJorinn~poJis
Tubnr,,o Farmacologia SC Brasif
BINDING OF FE” AND PREFERENTIAL CLEAVAGE OF DNA. Timothy Cole, Priya Rai, Ernst Henle, Stuart Linn. De artment of Molecular and Cell Biolgy, University of California at B erkeley.
e Cifncias
Considering both antioxidant and prooxidant activities of caffeic acid (CA) in vitro assays, the aim of this study was to investigate these effects in viva. A group of Male albine Wistar rats received only DMSO 10% and other three test groups received different doses (0.009,0.03 e 0.09 mmol/Kg) of CA in suspension (DMSO 10%) intraperitoneally. The plasma oxidation was evaluated through the tiobarbituric acid reactive substances (TBARs) level. The rats treated with different doses of CA showed significantly lower TBARs level in a dose-dependent way. The plasma antioxidant capacity was verified by the protection against oxidative damages to desoxyribose induced by Fe3+-ascorbate-H202. The plasma of animals treated with different doses of CA was able to inhibit desoxyribose degradation. The level of lipid peroxidation in the liver was determined by TBARs level. ln lower doses (0.009 and 0.003 mmol/kg) it was observed a tendency to increase lipid peroxidation levels in liver, when compared with non-treated group. Nevertheless, in the higher dose a significant decreased of TBARs levels was observed if compared with the dose 0.03 mmol/kg, but not with the control. In order to evaluate the genotoxic effects of CA the Comet assay was used. This study demonstrated that in the higher doses CA was able to increase the DNA damage, by increasing the total class of damage. In addition, it was observed that only class the II DNA damage was increased significantly in the group treated with higher dose. These results demonstrated that CA possess paradoxical effects in viva, as reported in the literature. Therefore more studies about antioxidant and prooxidant mechanisms of this phenolic acid and other related compounds in vitro is necessary. Support: CNPq
The DNA sequence RTGR (R = purine) has been shown to undergo preferential cleavage by Fe”/H202 at the sugar moiety of the thymidine. This sequence 1s a required motif in the regulatory element of many genes that respond to oxygen and iron stress. We proposed that enhanced cleavage results from stronger binding of iron to this particular DNA sequence. Computer modelling of the interactions between Fe” and B-form DNA containing ATGA showed that iron could be octahedrally coordinated within this site. As a result of these initial findings, steady state fluorescence and NMR studies of a DNA 16-mer containing ATGA were performed. 2-Amino-purine (2.AP) was substituted for adenine at various sites within the duplex and fluorescence was monitored. Significant variations in solvent exposure and strength of stacking interactionsof 2-AP at the different sites were found in the absence of Fe’ These variations were further evidenced by differences in fluorescence intensity of 2-AP within the site and at flanking upon addition of Fe”. The observed differences also positions correlated with the degree of change in fluorescence intensity caused by localization of iron to these sites during titration with acrylamide, a quenching agent. NMR spectra showed that the protons of conventional bases experienced variable amounts of line broadening upon binding of iron. We propose that the native structure of ATGA within duplex DNA is particularly attractive for Fe” binding, resulting in preferentialcleavage upon addition of H,O, and possibly its utilization as a promoter element.
I 282 IDENTIFICATION OF OXIDIZED PROTEIN HYDROLASE IN HUMAN ERYTHROCYTES Tomofumi Fuiino. Kazuomi Watanabe, Masatoshi Beppu, Kiyomi Kikugawa, and Hideyo Yasuda, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachiqji, Tokyo, Japan We purified and characterized an 8OkDa serine protease in human erythrocytes which degrade oxidized membrane proteins. The enzyme was named as oxidized protein hydrolase (OPH). OPH is found to be present in cytosol and become adherent to membranes upon cell oxidation and have high selectivity for proteins modified by oxidation and glycation. We determined partial amino acid sequence of OPH. The N-terminal amino acid of OPH was suggested to be masked. Six peptide fragments obtained by digestion of OPH with lysyl endopeptidase were isolated by use of reverse-phase high performance liquid chromatography, and the Nterminal sequence of each peptide was determined. Results of homology search of amino acid sequence of each peptide strongly suggested that OPH was identical with human liver acyl peptide hydrolase (ACPH). OPH showed ACPH activity Glutathione Stransferase (GST)-tagged recombinant ACPH (rACPH) was prepared from cDNA of human erythroleukemic cell line K-562. rACPH reacted with anti-OPH antiserum from rabbit. rACPH showed OPH activity when hydrogen peroxide-oxidized or glycated bovine serum albumin was used as substrates. As well as the enzyme activities of OPH, those of rACPH were inhibited by diisopropyl fluorophosphate. The results clearly demonstrate that ACPH whose physiological function has not yet been well characterized can play an important role as OPH in destroying oxidatively damaged proteins in living cells.
OXYGEN
MOLECULAR CHLORINE GENERATED BY MYELOPEROXIDASE PRODUCES 5-CHLOROCYTOSINE: IMPLICATIONS FOR CANCER E Henderson. ~aeman Byun, and Jny W. Heinecke. St. Louis, MO 63110.
k&%&,
Wnskmgton
Oxidation of nucleic acids has been implicated as a mode of cytotoxicity and mutagenicity at sites of inflammation. We now demonstrate a novel pathway for chlorination of cytosine by myeloperoxidase, a heme protein secreted by phagocytes. A screen of nucleoside reactivity with the myeloperoxidase-H202-C1system revealed that deoxycytidine was a major substrate for oxidation. The product was identified as 5-chloro-2’-deoxycytidine using HPLC, gas chromatography/mass spectrometry, electrospray mass spectrometry, and high resolution NMR. Chlorination by HOCl, an oxidant generated by myeloperoxidase, required chloride and H+, implicating molecular chlorine (C12) as the actual halogenating species. Activated human neutrophils generated 5-chloro-2’-deoxycytidine in a reaction that was blocked by catalase and peroxidase inhibitors. These observations support a role for myeloperoxidase in oxidizing nucleic acids at sites of inflammation. Furthermore, this raises the possibility that halogenated nucleobases play a role in cytotoxicity and the pathogenesis of cancer.
I
9
9
s93
OF ANTIOXIDANT,
PROOXIDANT
AND
GENOTOXIC EFFECTS OF CAFFEIC ACID: A IN VIVO STUDY Ka I n Ckless. Michelle Susin, Ryane Hess, Cl.udm Simm, Rosa Ri&&-do-V& Fnrmncfuticns
Fnrmcin UNJSUL UFSC FJorinn~poJis
Tubnr,,o Farmacologia SC Brasif
BINDING OF FE” AND PREFERENTIAL CLEAVAGE OF DNA. Timothy Cole, Priya Rai, Ernst Henle, Stuart Linn. De artment of Molecular and Cell Biolgy, University of California at B erkeley.
e Cifncias
Considering both antioxidant and prooxidant activities of caffeic acid (CA) in vitro assays, the aim of this study was to investigate these effects in viva. A group of Male albine Wistar rats received only DMSO 10% and other three test groups received different doses (0.009,0.03 e 0.09 mmol/Kg) of CA in suspension (DMSO 10%) intraperitoneally. The plasma oxidation was evaluated through the tiobarbituric acid reactive substances (TBARs) level. The rats treated with different doses of CA showed significantly lower TBARs level in a dose-dependent way. The plasma antioxidant capacity was verified by the protection against oxidative damages to desoxyribose induced by Fe3+-ascorbate-H202. The plasma of animals treated with different doses of CA was able to inhibit desoxyribose degradation. The level of lipid peroxidation in the liver was determined by TBARs level. ln lower doses (0.009 and 0.003 mmol/kg) it was observed a tendency to increase lipid peroxidation levels in liver, when compared with non-treated group. Nevertheless, in the higher dose a significant decreased of TBARs levels was observed if compared with the dose 0.03 mmol/kg, but not with the control. In order to evaluate the genotoxic effects of CA the Comet assay was used. This study demonstrated that in the higher doses CA was able to increase the DNA damage, by increasing the total class of damage. In addition, it was observed that only class the II DNA damage was increased significantly in the group treated with higher dose. These results demonstrated that CA possess paradoxical effects in viva, as reported in the literature. Therefore more studies about antioxidant and prooxidant mechanisms of this phenolic acid and other related compounds in vitro is necessary. Support: CNPq
The DNA sequence RTGR (R = purine) has been shown to undergo preferential cleavage by Fe”/H202 at the sugar moiety of the thymidine. This sequence 1s a required motif in the regulatory element of many genes that respond to oxygen and iron stress. We proposed that enhanced cleavage results from stronger binding of iron to this particular DNA sequence. Computer modelling of the interactions between Fe” and B-form DNA containing ATGA showed that iron could be octahedrally coordinated within this site. As a result of these initial findings, steady state fluorescence and NMR studies of a DNA 16-mer containing ATGA were performed. 2-Amino-purine (2.AP) was substituted for adenine at various sites within the duplex and fluorescence was monitored. Significant variations in solvent exposure and strength of stacking interactionsof 2-AP at the different sites were found in the absence of Fe’ These variations were further evidenced by differences in fluorescence intensity of 2-AP within the site and at flanking upon addition of Fe”. The observed differences also positions correlated with the degree of change in fluorescence intensity caused by localization of iron to these sites during titration with acrylamide, a quenching agent. NMR spectra showed that the protons of conventional bases experienced variable amounts of line broadening upon binding of iron. We propose that the native structure of ATGA within duplex DNA is particularly attractive for Fe” binding, resulting in preferentialcleavage upon addition of H,O, and possibly its utilization as a promoter element.
I 282 IDENTIFICATION OF OXIDIZED PROTEIN HYDROLASE IN HUMAN ERYTHROCYTES Tomofumi Fuiino. Kazuomi Watanabe, Masatoshi Beppu, Kiyomi Kikugawa, and Hideyo Yasuda, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachiqji, Tokyo, Japan We purified and characterized an 8OkDa serine protease in human erythrocytes which degrade oxidized membrane proteins. The enzyme was named as oxidized protein hydrolase (OPH). OPH is found to be present in cytosol and become adherent to membranes upon cell oxidation and have high selectivity for proteins modified by oxidation and glycation. We determined partial amino acid sequence of OPH. The N-terminal amino acid of OPH was suggested to be masked. Six peptide fragments obtained by digestion of OPH with lysyl endopeptidase were isolated by use of reverse-phase high performance liquid chromatography, and the Nterminal sequence of each peptide was determined. Results of homology search of amino acid sequence of each peptide strongly suggested that OPH was identical with human liver acyl peptide hydrolase (ACPH). OPH showed ACPH activity Glutathione Stransferase (GST)-tagged recombinant ACPH (rACPH) was prepared from cDNA of human erythroleukemic cell line K-562. rACPH reacted with anti-OPH antiserum from rabbit. rACPH showed OPH activity when hydrogen peroxide-oxidized or glycated bovine serum albumin was used as substrates. As well as the enzyme activities of OPH, those of rACPH were inhibited by diisopropyl fluorophosphate. The results clearly demonstrate that ACPH whose physiological function has not yet been well characterized can play an important role as OPH in destroying oxidatively damaged proteins in living cells.
OXYGEN
MOLECULAR CHLORINE GENERATED BY MYELOPEROXIDASE PRODUCES 5-CHLOROCYTOSINE: IMPLICATIONS FOR CANCER E Henderson. ~aeman Byun, and Jny W. Heinecke. St. Louis, MO 63110.
k&%&,
Wnskmgton
Oxidation of nucleic acids has been implicated as a mode of cytotoxicity and mutagenicity at sites of inflammation. We now demonstrate a novel pathway for chlorination of cytosine by myeloperoxidase, a heme protein secreted by phagocytes. A screen of nucleoside reactivity with the myeloperoxidase-H202-C1system revealed that deoxycytidine was a major substrate for oxidation. The product was identified as 5-chloro-2’-deoxycytidine using HPLC, gas chromatography/mass spectrometry, electrospray mass spectrometry, and high resolution NMR. Chlorination by HOCl, an oxidant generated by myeloperoxidase, required chloride and H+, implicating molecular chlorine (C12) as the actual halogenating species. Activated human neutrophils generated 5-chloro-2’-deoxycytidine in a reaction that was blocked by catalase and peroxidase inhibitors. These observations support a role for myeloperoxidase in oxidizing nucleic acids at sites of inflammation. Furthermore, this raises the possibility that halogenated nucleobases play a role in cytotoxicity and the pathogenesis of cancer.
I
9
9
s93