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Page 1: Role of iron ion chelation by quinones in their reduction, OH-radical generation and lipid peroxidation

Session 7: Oxidative Mechanisms of Cell “Death” 523

7~29

7:31

ZONE-SPECIFIC PARADOXICAL OXIDATIVE INJURY IN HYPOPERFUSED RAT LIVER: ASSESSMENT BY DIGITAL MICROFLUOROGRAPHY H. Suzuki, M. Suematsu, H. Ishii, S. Kato, Y. Ishimura, and M. Tsuchiya. Departments of Biochemistry and Internal Medicine, Keio University, Tokyo

Pathological relevance of reductive stress in zone-specific oxidative cell injury was investigated in isolated hypoperfused rat liver. The intralobular heterogeneity of the redox state, mitochondrial dysfunction and intracellular hydroperoxide formation were visually monitored by digital microfluorography of NADH autofluorescence (I), rhodamine I23 (2) and dichlorofluorescein (DCF) fluorescence(3), respectively Cell viability was assessed by propidium iodide (PI) method combined with other fluorochromes (2.3). In another set of experiments, effects of prostaglandin El on oxidative cell injury were also studied. Under the 25% low-flow perfusion, NADH increased time-dependently and reached a steady state at 10 min among the entire lobules. The depletion of mitochondrial membrane uotential reached more than 20 mV in all portions of the lobules at 60 min. The onset of oxidative cell iniury as judged by DCF and PI was observed at 40 min in the marginally oxygenated proximal portion of anoxic pericentral regions, and the oxidative impact reached a maximum level at 60 min. Pretreatment with prostaglandin El not only attenuated early NADH elevation and mitochondrial dysfunction induced by hypoperfusion but also diminished subsequent midzonal oxidative injury. Since prostaglandin El has no oxyradical-scavenging action, the preventive effect of this reagent on the hypoxia-induced oxidative cell injury is concluded to be a consequence of the attenuation of mitochondrial dysfunction. These results suggest that, in low-flow hypoxia, early reductive stress plays a key role in the initiation of midzonal oxidative changes which may lead to subsequent centrilobular necrosis. References: (I) Microvasc Res (in press) (2) Lab Invest 67, 434, 1992. (3) Gastroenterology 103, 994, 1992.

ROLE OF IRON ION CHELATION BY QUINONES IN THEIR REDUCTION, OH-BADICAL GENERA- TION AND LIPID PEROXIDATION Dikalov S.+, Alov P. and Rangelova D. *Biophysioxtl group, Institute of Chardcal Kinetics & Combustion, 630090 Novosibirsk, Russia, Institute of PhyGology, SC& Bolgaria.

To study the role of the complex of quinonen with iron ions in the processes of quinone reduction and OH- radical generation in the presence of aecorbate (AH) and glutathione (GSH) the quinone-chelatora have been used: 2-

phenyl-4-butykninonaphtho [2,3-h] quinolin&one-7,12 (Qe)

and adknycin (ti). 2-phenyl-S-nitronaphtho[2,3-g] indo- dione+ 1 l(Qn),2-(3-hydroxypropyl)anthmquinone (AQOP)

and 24imethylamino-3-c&r-1,4-naphthoquinone (DCNQ)

were chosen as quinonea that do not chelate iron ions. It was found that, unlike to A& and quinonea Qn, AQOP, andDCNQ,additionofQctoAHandGSHleadstosemi- quinone EPR spectrum formation and OH-radical genera- tion via the complex of Qc with iron ions. It was demonat- rated that all these quinones can be reduced by AH. How- ever reduction wmtant of Qc-Fe(3+) by the AH was 9829 M-W, while DCNQ reduction constant was only 0.042 M-l c-1. It was found that in the presence of GSH only comp- lexes of Qc and A& with iron m reduced It is con&led

that the capability of Qc to reduce and to generate -OH is related to intramolecular electron &an&r: Fe(Z+)-Qc

aFe(3+m. The capability of Qc to generate oxygen radicals and to inhibite lipid peroxidation may be interesting for desig& of quinone_containins ant&iotics.

ROLE OF REACTIVE OXIDANTS IN LETHAL CELL INJURY 7:30 CAUSED BY ENERGY DEPLETiON Frank F. Sun, Bruce M. Taylor and William E. Fleming The Upjohn Company, Kalamazoo, MI. 49001 USA

Reactive oxidants have been proposed as important mediators in ischemia reperfusion injury to brain cells. The present study examined the formation of reactive oxygen species in cultured human astrocytoma cells UC-l IMG which have been severely injured by energy depletion. Exposure of cultured astrocytoma cells to 75 uM of sodium iodoacetate (IAA) caused a rapid decrease of intracellular ATP and loss of viability. We monitored the formation of reactive oxygen species (ROS) in the injured cells using the fluorescent probe 2’,7’-dichlorofluorescin-diacetate (DCF-DA) and an ACAS 570 confocal cytometer. A general membrane probe (PKH-26) and other organelle specific markers were used to localize the site of intracellular ROS. ROS fluorescence was observed throughout the entire injured cell with a greater intensity evident in and around the nucleus. When the DCF fluorescence was quantified in a fluorescence plate reader, we found that the level of ROS in IAA treated astrocytoma cells was not evident for the first 30 minutes post IAA after which it continually increased over a 4 hour incubation. The levels of ROS fluorescence in the IAA injured cells were comparable to those cells irreversible damaged by millimolar concentrations of H,O, or t-butyl hydroperoxide. However, dose response studies showed that the elevation of intracellular ROS did not necessarily lead to cell killing. The IAA induced increases in ROS could be blocked by common antioxidants and the ability of these antioxidants to abolish intracellular ROS accumulation roughly corresponds to their ability to delay lethal injury in these cells. We conclude that intracellular ROS is an important contributor but not an essential element that causes irreversible cell injury

GENERATION OF OXYGEN RADICALS IN THE 7:32 CUL.TUREDPLANTcELLs S.Dikalo+, A.Dikalova, E.Deyneko and R.Salganik Institute of Cytology & Genetica and *Institute of Chemi- cal Kinetics & Combustion, 630090 Novo&ir& Rusk

Agrowingbodyofevidenceindicatesthatinthe cultured plant cells numerous mutationa arise which are ma&&ted in the regenerated plat&. Thin phenomenon is known as somaclonal variability. However the nature of mutagenic factors is not clear. It was reason&le to suggest that mutations in plant cell8 are induced by nonnamml streW%l condition8 of ar&ial medium. The formation of oxygen radicals can be Wengthened by the iron -1% Fe-EDTA, which is usually added to the cell medium. To study the validity of these sugge6tions we monitored the concentrationoffreeradicalsinplanteecdlingAcallueand cell suspension of Medicago. It was found that generation of OH-radicals in cell euepeneion and in calh~ is much higherthanin!%[email protected]&ra- tion of such iron complexes a8 Fe-ADP, Fe&rate, Fe- deferoxamine which inhiited the oxygen radicala forma- tion increased the velocity of the calhw and euapen&m cul- turea growth, and decreases the amount of necroaes. The increase of the OH-radicals form&m was followed by the decrease of c&al&K activity. Intenk realIangements of mitochondrial DNA is observed in cult&ted plant cella.

The data obtained support the notion that cult&at@ of cells induce OH-radicals generation and the latter act as inducti of mutations, growth retardation and necrm~~