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Activities of free radical scavengers in tests designed to detect lipid peroxyl or superoxide radical scavenging
ACTIVITIES OF FREE RADICAL SCAVENGERS IN TESTS DESIGNED TO DETECT LIPID PEROXYL OR SUPEROXIDE R A D I C A L SCAVENGING. C.Carter, O.Curet and A. Deffois. Synthelabo Recherche, 10 Rue des Carrieres, BP248, 92504 RueiI-Malmaison Cedex, France.
INDUCTION OF DAUNORUBICIN CARBONYL REDUCING ENZYMES IN CARCINOMA CELLS M. Soldan, K.J. Netter and E. Maser Dept. of Pharmacology & Toxicology, Philipps-University, Karl-von Frisch-Strasse 1, 35033 Marburg, Germany A major problem in cytostatic treatment of malignant tumors is the development of chemoresistant cell clones. The phenomenon of multidrug resistance in these tumor cells is usually related to the overexpression of a plasma membrane glycoprotein (P-170), which mediates enhanced drug efflux and prevents intracellular drug accumulation. It is known that P-170 cannot fully account for chemoresistance and that enzymatic mechanisms like glutathion transferase and glucuronyl transferase also contribute to multidrug resistance. Anthracyclines like daunorubicin, are the most valuable cytostatic agents in clinical use, but their usefulness is limited by tumor cell drug resistance. Carbonyl reduction of daunorubicin leads to 13-hydroxydaunorubicinol, the major metabolite with a significant lower antineoplastic potency compared to the parent drug. In the present study we cultured two pancreas carcinoma cell lines (a sensitive parental line and its daunorubicin resistant subline) in the present of increasing concentrations of daunorubicin (DRC) and demonstrate that DRC treatment itself leads to an enhanced expression of DRC carbonyl reducing enzymes in subcellular fractions of both cell lines. The principal difference between the two cell lines becomes most apparent at high dose DRC supplementation, at which the resistant cells exhibited highest inducibility of DRC inactivating enzymes, whereas respective sensitive cells already showed an impairment of cellular viability. The results suggest that inactivation of anthracyclines by carbonyl reduction has to be considered as one of the enzymatic mechanisms which might contibute to the acquired resistance towards these drug.
We have tested the activities of various compounds in relation to their abilities to prevent spontaneous lipid peroxidation of brain membranes, to quench the diphenylpicrylhydrazine (DPPH) radical, or to prevent the oxidation of cytochrome C (cyt C) or nitroblue tetrazolium (NBT) by xanthine/xanthine oxidase superoxide generation. Suspected hydroxyl radical scavengers including c~-t-phenyl-butyl nitrone and related spin traps, mannitol, salicylate or DMSO had little effect at mM concentrations in any test. Lipid peroxidation was inhibited by (ICsopM) phenoxazine (0.01), diphenylphenylenediamine (0.05) gallic acid (0.24), ellagic acid (0.52), tocopherol (1), fraxetine (2), butylated hydroxytoluene (2) , promethazine (3.1) and tirilazad (159). The same compounds quenched the DPPH radical generally with lower but correlated potency. Of the active compounds, none except (IC~pM) phenoxazine (NBT, 11.7; cyt C >1000) fraxetine (cyt C, 0.07; NBT= 0.1), gallic acid (NBT, 1.3pM) and ellagic acid (NBT 4.2pM) displayed activity in the NBT or Cyt C tests at 1000pM. These compounds showdiffering profiles accross a battery of radical scavenging tests t h a t may be related either to their accessibility to the site of radical generation or to a selectivity of action versus differentspecies of radical or elements involved in free radical generation.~ The relevance of their selectivity of action in relation to more functional tests related to free radical damage remains to be assessed.
THE PINEAL HORMONE MELATONIN INHIBITS LPSINDUCED LIPID PEROXIDATION I N VITRO E. Sewerynek, D. Melchiorri, LD.Chen and RJ. Reiter Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284 The protective effect of melatonin against lipopolysaccharide (LPS)-induced oxidative damage was examined i n vitro. Lung, liver and brain malonaldehyde (MDA) plus 4-hydroxyalkenals (4HDA) concentration was measured as an index of lipid peroxidation. Homogenates &brain, lung and liver were incubated with LPS at concentrations &either 1, 10, 50, 200 or 400 ~g/ml for 1 h and, in another study, LPS (400 gg/ml) for either 15, 30 or 60 rain. Melatonin at increasing concentrations from 0.01-3 mM either alone or together with LPS (400 gg/ml) was used. Liver, brain and lung lipid peroxidation increased after LPS at concentrations of 10, 50; 200 or 400 gg/ml; this effect was concentration-dependent. The highest levels of lipid peroxidation products were observed after incubation with an LPS concentration of 400 ~tg/ml for 60 min; in liver and lung this effect was totally suppressed by.melatonin and partially suppressed in brain in a concentration-dependent manner. Additionally, melatonin alone was effective in brain at concentrations of 0.1 to 3 mM, in lung at 2 to 3 mM and in liver at 0.1 to 3 raM; in all tissues, the inhibitory effects of melatonin on lipid peroxidation were always directly correlated with the concentration on melatonin in the medium. The results show that the direct effect of LPS on the lipid peroxidation following endotoxin exposure is markedly reduced by melatonin. Acknowledgments: ES (Fogarty Fellowship - F05 TWO 5014-01), research (NSF #91-21263).
EVIDENCE FOR A DISSIMILAR MECHANISM OF ENHANCEMENT OF INORGANIC OR ORGANIC HYDROPEROXIDE CYTOTOXICITY BY L-HISTIDINE.
A. Guidarelli, L Palomba, P. Sestili, F. Cattabeni and O. CantoN. lstituto di Farmacologia e Farmacognosia and Centro di Farmacologia Oncologica Sperimentale, Universit& di Urbino (Italy). The amino acid L-Histidine markedly increases hydroperoxide-induced eytotoxicity and DNA single strand breakage in CHO cells. The enhancing effect was greater for the organic peroxides, cumene hydroperoxide (CuOOH) and tert-butyl hydroperoxide (tB-OOH), as compared to hydrogen peroxide (H202). Among the organic peroxides, the level of enhancement of the cytotoxic response was higher in cells exposed to tB-OOH, whereas the increased production of DNA single strand breaks (SSBs) was more pronounced in cells treated with Cu-OOH. Surprisingly, the activation of the enzyme poly(ADP-ribose)polymerase, as measured by a 3-aminobenzamide-inhibitable decline in NAD levels, was restricted to cells treated with H202, with or without L-Histidine since no reduction of NAD was observed following treatment with the organic peroxides, even under conditions of accumulation of very high levels of DNA SSBs (e.g. in the presence~ of L-Histidine). The membrane-permeant iron chelating agent o-phenanthroline was found to protect the cells against killing and DNA SSB-formation elicited by all the hydroperoxides, both in the absence or presence of the amino acid, suggesting that the metal plays a pivotal role in the induction of lethality, as well as DNA SSBs, irrespectively of whether L-Histidine was absent or present during treatment with the peroxides. In contrast, the antioxidant DPPD was unable to afford protection under any of the above experimental conditions, indicating that peroxidative membrane processes are not involved in the induction of CHO cell killing and production of DNA SSBs. An important event triggered by the amino acid in cells treated with H202 is the formation of DNA double strand breaks (DSBs), a type of lesion considered to be highly cytotoxic which however was not detected in cells exposed to L-Histidine and the organic peroxides. It is important to note that a large body of evidence indicates that DNA DSBs mediate the cytotoxic response in ceils challenged with the cocktail H202 - LHistidine. Thus, L-Histidine enhances a number of deleterious effects in cells exposed to H202, tB-OOH or Cu-OOH although the mechanism for the enhancement of the cytotoxic response appears to be different for inorganic or organic hydroperoxides.
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140
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INDUCTION OF DAUNORUBICIN CARBONYL REDUCING ENZYMES IN CARCINOMA CELLS M. Soldan, K.J. Netter and E. Maser Dept. of Pharmacology & Toxicology, Philipps-University, Karl-von Frisch-Strasse 1, 35033 Marburg, Germany A major problem in cytostatic treatment of malignant tumors is the development of chemoresistant cell clones. The phenomenon of multidrug resistance in these tumor cells is usually related to the overexpression of a plasma membrane glycoprotein (P-170), which mediates enhanced drug efflux and prevents intracellular drug accumulation. It is known that P-170 cannot fully account for chemoresistance and that enzymatic mechanisms like glutathion transferase and glucuronyl transferase also contribute to multidrug resistance. Anthracyclines like daunorubicin, are the most valuable cytostatic agents in clinical use, but their usefulness is limited by tumor cell drug resistance. Carbonyl reduction of daunorubicin leads to 13-hydroxydaunorubicinol, the major metabolite with a significant lower antineoplastic potency compared to the parent drug. In the present study we cultured two pancreas carcinoma cell lines (a sensitive parental line and its daunorubicin resistant subline) in the present of increasing concentrations of daunorubicin (DRC) and demonstrate that DRC treatment itself leads to an enhanced expression of DRC carbonyl reducing enzymes in subcellular fractions of both cell lines. The principal difference between the two cell lines becomes most apparent at high dose DRC supplementation, at which the resistant cells exhibited highest inducibility of DRC inactivating enzymes, whereas respective sensitive cells already showed an impairment of cellular viability. The results suggest that inactivation of anthracyclines by carbonyl reduction has to be considered as one of the enzymatic mechanisms which might contibute to the acquired resistance towards these drug.
We have tested the activities of various compounds in relation to their abilities to prevent spontaneous lipid peroxidation of brain membranes, to quench the diphenylpicrylhydrazine (DPPH) radical, or to prevent the oxidation of cytochrome C (cyt C) or nitroblue tetrazolium (NBT) by xanthine/xanthine oxidase superoxide generation. Suspected hydroxyl radical scavengers including c~-t-phenyl-butyl nitrone and related spin traps, mannitol, salicylate or DMSO had little effect at mM concentrations in any test. Lipid peroxidation was inhibited by (ICsopM) phenoxazine (0.01), diphenylphenylenediamine (0.05) gallic acid (0.24), ellagic acid (0.52), tocopherol (1), fraxetine (2), butylated hydroxytoluene (2) , promethazine (3.1) and tirilazad (159). The same compounds quenched the DPPH radical generally with lower but correlated potency. Of the active compounds, none except (IC~pM) phenoxazine (NBT, 11.7; cyt C >1000) fraxetine (cyt C, 0.07; NBT= 0.1), gallic acid (NBT, 1.3pM) and ellagic acid (NBT 4.2pM) displayed activity in the NBT or Cyt C tests at 1000pM. These compounds showdiffering profiles accross a battery of radical scavenging tests t h a t may be related either to their accessibility to the site of radical generation or to a selectivity of action versus differentspecies of radical or elements involved in free radical generation.~ The relevance of their selectivity of action in relation to more functional tests related to free radical damage remains to be assessed.
THE PINEAL HORMONE MELATONIN INHIBITS LPSINDUCED LIPID PEROXIDATION I N VITRO E. Sewerynek, D. Melchiorri, LD.Chen and RJ. Reiter Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284 The protective effect of melatonin against lipopolysaccharide (LPS)-induced oxidative damage was examined i n vitro. Lung, liver and brain malonaldehyde (MDA) plus 4-hydroxyalkenals (4HDA) concentration was measured as an index of lipid peroxidation. Homogenates &brain, lung and liver were incubated with LPS at concentrations &either 1, 10, 50, 200 or 400 ~g/ml for 1 h and, in another study, LPS (400 gg/ml) for either 15, 30 or 60 rain. Melatonin at increasing concentrations from 0.01-3 mM either alone or together with LPS (400 gg/ml) was used. Liver, brain and lung lipid peroxidation increased after LPS at concentrations of 10, 50; 200 or 400 gg/ml; this effect was concentration-dependent. The highest levels of lipid peroxidation products were observed after incubation with an LPS concentration of 400 ~tg/ml for 60 min; in liver and lung this effect was totally suppressed by.melatonin and partially suppressed in brain in a concentration-dependent manner. Additionally, melatonin alone was effective in brain at concentrations of 0.1 to 3 mM, in lung at 2 to 3 mM and in liver at 0.1 to 3 raM; in all tissues, the inhibitory effects of melatonin on lipid peroxidation were always directly correlated with the concentration on melatonin in the medium. The results show that the direct effect of LPS on the lipid peroxidation following endotoxin exposure is markedly reduced by melatonin. Acknowledgments: ES (Fogarty Fellowship - F05 TWO 5014-01), research (NSF #91-21263).
EVIDENCE FOR A DISSIMILAR MECHANISM OF ENHANCEMENT OF INORGANIC OR ORGANIC HYDROPEROXIDE CYTOTOXICITY BY L-HISTIDINE.
A. Guidarelli, L Palomba, P. Sestili, F. Cattabeni and O. CantoN. lstituto di Farmacologia e Farmacognosia and Centro di Farmacologia Oncologica Sperimentale, Universit& di Urbino (Italy). The amino acid L-Histidine markedly increases hydroperoxide-induced eytotoxicity and DNA single strand breakage in CHO cells. The enhancing effect was greater for the organic peroxides, cumene hydroperoxide (CuOOH) and tert-butyl hydroperoxide (tB-OOH), as compared to hydrogen peroxide (H202). Among the organic peroxides, the level of enhancement of the cytotoxic response was higher in cells exposed to tB-OOH, whereas the increased production of DNA single strand breaks (SSBs) was more pronounced in cells treated with Cu-OOH. Surprisingly, the activation of the enzyme poly(ADP-ribose)polymerase, as measured by a 3-aminobenzamide-inhibitable decline in NAD levels, was restricted to cells treated with H202, with or without L-Histidine since no reduction of NAD was observed following treatment with the organic peroxides, even under conditions of accumulation of very high levels of DNA SSBs (e.g. in the presence~ of L-Histidine). The membrane-permeant iron chelating agent o-phenanthroline was found to protect the cells against killing and DNA SSB-formation elicited by all the hydroperoxides, both in the absence or presence of the amino acid, suggesting that the metal plays a pivotal role in the induction of lethality, as well as DNA SSBs, irrespectively of whether L-Histidine was absent or present during treatment with the peroxides. In contrast, the antioxidant DPPD was unable to afford protection under any of the above experimental conditions, indicating that peroxidative membrane processes are not involved in the induction of CHO cell killing and production of DNA SSBs. An important event triggered by the amino acid in cells treated with H202 is the formation of DNA double strand breaks (DSBs), a type of lesion considered to be highly cytotoxic which however was not detected in cells exposed to L-Histidine and the organic peroxides. It is important to note that a large body of evidence indicates that DNA DSBs mediate the cytotoxic response in ceils challenged with the cocktail H202 - LHistidine. Thus, L-Histidine enhances a number of deleterious effects in cells exposed to H202, tB-OOH or Cu-OOH although the mechanism for the enhancement of the cytotoxic response appears to be different for inorganic or organic hydroperoxides.
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140
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