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Paradoxical effects of 1,10-phenanthroline on metal-mediated and free radical-induced cytotoxicity
El
1
PARADOXICAL EFFECTS OF l,lO-PHENANTHROLINE METAL-MEDIATED AND FREE RADICAL-INDUCED CYTOTOXICITY Ben-Zhan &and Mordechat Hebrew Universzty-Hadassnh ferusnlem 91120, lsrael
ON
Chevion. Department o Cellular Biochemistry, Schools ofMedicine an df Dental Medicine,
In this report, we will present the paradoxical effects of l,lO-phenanthroline (OF) on metal-mediated and free radical-induced cytotoxicity in two different systems. 1). The effect OP on copper-mediated cytotoxicity of pentachlorophenol (WI’, a widely used wood preservative and a ubiquitous environmental pollutant) was found to be dependent on the molar ratio (R) of [OP]/[Cu(II)]: the bacterial killing was enhanced as R values increased, then reaching a maximum, further increase of R values led to a progressive decrease of this enhancement. A lipophilic ternary complex was formed between OP, Cu(I1) and PCP at low R values, while at high R values this ternary complex disappears. Therefore, the formation and decay of the lipophilic ternary complex might be responsible for the paradoxical cytotoxic effect. 2). Interestingly, the effect of OF on H202-mediated toxicity to E. coli was also dependent on OP concentrations. At low concentrations, OP markedly enhanced H202-induced bacterial killing but shifted to protection at high concentrations. This was correlated with the effect of OF on iron-mediated and H202-induced DNA damage. It is known that iron and OP could form three complexes. The mono and bis complexes could react with H202 to produce OH radicals, whereas the tris complex is stable towards H202. Therefore, the enhancing effect at low OP concentration could be explained by the reaction of OF with intracellular Fe(B), i.e., the mono and bis complexes are more reactive than intracellular Fe(II), while the protective effect at high OP concentration could be due to the exclusive formation of the tris complex. Based on these results, we suggest that care should be taken when employing OF to probe the role of transition metal ions in biological systems.
I
39
EVIDENCE FOR THE CONTAMINATION REDOX-ACTIVE METALS IN COMMERCIALLY-AVAILABLE PHYTIC Ben-Zhnn Zhu and Mordechai Hebrew University-Hadassah lerusalem 91120, Israel
I
We reported hepatic mitochondrial dysfunction in Avian Pulmonary Hypertension Syndmme (PHS) (Poultry Sci. 73114. 1999); a costly metabolic disease in meat chickens (broilers). Blood oxygenation problems and inefficient oxygen utilization at the cellular level characterize this disease. The major goal of this study was to identify specific defects in O2 use by the electron transport chain (ETC) of PHS hepatic mitochondna (Mito). Mito were isolated from broilers with and without PHS. The primary indices of Mite function, the respiratory control ratio (RCR) and the ADP/O were determined by monitoring 02 consumption, with and without additions of site specific respiratory inhibitors (e.g. antimycin, rotenone, malonate, thenoyttrtfluoroacetone jTTFA]. myxothiazol, KCN). The RCR is State 3 respiration (ADP excess) divided by State 4 (ADP depleted). The ADP/O is ADP added divided by 02 consumed during State 3. When State 3 was inhibited by malonate or TTFA, (which block ETC respiration at Complex II); higher O2 consumption remained in PHS Mto than in COntmls. These findings suggest that there is greater electron leakage at Complex II in the ETC of PHS Mito. Lower levels of Vit E and GSH in PHS Mito indicate mitochondrtal oxidative stress contributes to the pathophysiology of PHS. Interestingly, sequential additions of ADP increased the ADP/O ratio in Controls, but not in PHS Mito. Also, the RCR improved in both groups, but the RCR was higher in Controls than in PHS Mito due to a greater decrease in State 4 respiration following sequential additions of ADP. These findings suggest that hepatic mitochondrial dysfunction in PHS may be due to both electron leakage associated with Complex II and an impaired ability to reduce State 4 respiration.
OXYGEN
ACID
Chevion. Department o Cellular Biochemistry, Schools of Medicine an df Dental Medicine,
Phytic acid has been used as a unique chelator to study the role (involvement) of transition metals (especially iron) in free radical research. In this communication, we found that i). the rate of auto-oxidation of ascorbic acid was enhanced by commercially-available phytic acid dose-dependently; ii). the phytic acid-enhanced ascorbic acid autooxidation could be markedly prevented by DTI’A or by the pretreatment of phytic acid with Chelex resin or Coalbumin; iii). phytic acid inhibited iron-catalyzed but not copper-catalyzed ascorbic acid auto-oxidation, indicating copper-phytic acid complex is still redox active; and iv). the contamination with trace amounts of copper in commercially-available phytic acid was verified by Zeeman Atomic Absorption Spectrometer. Base on these findings, we recommend that 1). phytic acid should be pretreated by Chelex resin before use, and 2). high concentrations of phytic acid should be avoided, especially in free radical research.
Hepatic Mitochondrial Dyefunction and Electron Leak in Avian Pulmonary Hypertension Syndrome (PHS). David Cawthon and Walter Bottje. Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701
S24
OF
’ 9
9
1
PARADOXICAL EFFECTS OF l,lO-PHENANTHROLINE METAL-MEDIATED AND FREE RADICAL-INDUCED CYTOTOXICITY Ben-Zhan &and Mordechat Hebrew Universzty-Hadassnh ferusnlem 91120, lsrael
ON
Chevion. Department o Cellular Biochemistry, Schools ofMedicine an df Dental Medicine,
In this report, we will present the paradoxical effects of l,lO-phenanthroline (OF) on metal-mediated and free radical-induced cytotoxicity in two different systems. 1). The effect OP on copper-mediated cytotoxicity of pentachlorophenol (WI’, a widely used wood preservative and a ubiquitous environmental pollutant) was found to be dependent on the molar ratio (R) of [OP]/[Cu(II)]: the bacterial killing was enhanced as R values increased, then reaching a maximum, further increase of R values led to a progressive decrease of this enhancement. A lipophilic ternary complex was formed between OP, Cu(I1) and PCP at low R values, while at high R values this ternary complex disappears. Therefore, the formation and decay of the lipophilic ternary complex might be responsible for the paradoxical cytotoxic effect. 2). Interestingly, the effect of OF on H202-mediated toxicity to E. coli was also dependent on OP concentrations. At low concentrations, OP markedly enhanced H202-induced bacterial killing but shifted to protection at high concentrations. This was correlated with the effect of OF on iron-mediated and H202-induced DNA damage. It is known that iron and OP could form three complexes. The mono and bis complexes could react with H202 to produce OH radicals, whereas the tris complex is stable towards H202. Therefore, the enhancing effect at low OP concentration could be explained by the reaction of OF with intracellular Fe(B), i.e., the mono and bis complexes are more reactive than intracellular Fe(II), while the protective effect at high OP concentration could be due to the exclusive formation of the tris complex. Based on these results, we suggest that care should be taken when employing OF to probe the role of transition metal ions in biological systems.
I
39
EVIDENCE FOR THE CONTAMINATION REDOX-ACTIVE METALS IN COMMERCIALLY-AVAILABLE PHYTIC Ben-Zhnn Zhu and Mordechai Hebrew University-Hadassah lerusalem 91120, Israel
I
We reported hepatic mitochondrial dysfunction in Avian Pulmonary Hypertension Syndmme (PHS) (Poultry Sci. 73114. 1999); a costly metabolic disease in meat chickens (broilers). Blood oxygenation problems and inefficient oxygen utilization at the cellular level characterize this disease. The major goal of this study was to identify specific defects in O2 use by the electron transport chain (ETC) of PHS hepatic mitochondna (Mito). Mito were isolated from broilers with and without PHS. The primary indices of Mite function, the respiratory control ratio (RCR) and the ADP/O were determined by monitoring 02 consumption, with and without additions of site specific respiratory inhibitors (e.g. antimycin, rotenone, malonate, thenoyttrtfluoroacetone jTTFA]. myxothiazol, KCN). The RCR is State 3 respiration (ADP excess) divided by State 4 (ADP depleted). The ADP/O is ADP added divided by 02 consumed during State 3. When State 3 was inhibited by malonate or TTFA, (which block ETC respiration at Complex II); higher O2 consumption remained in PHS Mto than in COntmls. These findings suggest that there is greater electron leakage at Complex II in the ETC of PHS Mito. Lower levels of Vit E and GSH in PHS Mito indicate mitochondrtal oxidative stress contributes to the pathophysiology of PHS. Interestingly, sequential additions of ADP increased the ADP/O ratio in Controls, but not in PHS Mito. Also, the RCR improved in both groups, but the RCR was higher in Controls than in PHS Mito due to a greater decrease in State 4 respiration following sequential additions of ADP. These findings suggest that hepatic mitochondrial dysfunction in PHS may be due to both electron leakage associated with Complex II and an impaired ability to reduce State 4 respiration.
OXYGEN
ACID
Chevion. Department o Cellular Biochemistry, Schools of Medicine an df Dental Medicine,
Phytic acid has been used as a unique chelator to study the role (involvement) of transition metals (especially iron) in free radical research. In this communication, we found that i). the rate of auto-oxidation of ascorbic acid was enhanced by commercially-available phytic acid dose-dependently; ii). the phytic acid-enhanced ascorbic acid autooxidation could be markedly prevented by DTI’A or by the pretreatment of phytic acid with Chelex resin or Coalbumin; iii). phytic acid inhibited iron-catalyzed but not copper-catalyzed ascorbic acid auto-oxidation, indicating copper-phytic acid complex is still redox active; and iv). the contamination with trace amounts of copper in commercially-available phytic acid was verified by Zeeman Atomic Absorption Spectrometer. Base on these findings, we recommend that 1). phytic acid should be pretreated by Chelex resin before use, and 2). high concentrations of phytic acid should be avoided, especially in free radical research.
Hepatic Mitochondrial Dyefunction and Electron Leak in Avian Pulmonary Hypertension Syndrome (PHS). David Cawthon and Walter Bottje. Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701
S24
OF
’ 9
9