Toxicology Letters, 64/65 (19921547651 0 1992 Elsevier Science Publishers B.V., All rights reserved 03784274/92/$5.99

547

Role of reactive oxygen species in cell toxicity H. Sies and H. de Groot Institut fiirPhysiologische Chemie 1, Heinrich-Heine-Universitlit (Germany)

DiisseldorL Diisseldorf

Key words: Cytotoxicity; Reactive oxygen species; Nitric oxide; NADPH oxidase; Hypoxia-reoxygenation; Antioxidant depletion; NO synthase; Peroxynitrite; Chemiluminescence; Kupffer cells; a-Tocopherol; Carotenoids; Chromanoxyl radical; Glutathione; Ebselen; a-Tocopheroxyl radical

SUMMARY Several types of compound exert their cytotoxicity by generating reactive oxygen species, notably the superoxide anion radical. These include quinoid and nitroaromatic compounds serving as redox cyclers, i.e. producing super-oxide at the expense of NADPH and oxygen catalyzed by cellular reductases. In specialized cell-types employed in defense such as granulocytes, eosinophils and macrophages, myeloperoxidase, NADPH oxidase and nitric oxide synthase have been identified as major sources of reactive oxygen species in cell toxicity. These include hypochlorite, singlet oxygen, superoxide, nitric oxide and hydrogen peroxide. The interaction of superoxide and nitric oxide generates further oxidants such as peroxynitrite. Lumino-amplified chemiluminescence generated by Kupffer cells is partially sensitive to inhibitors of NO synthase. Superoxide dismutase has been found to catalyze a novel reaction, the reversible conversion of nitric oxide to the nitroxyl anion, the latter being viewed as another form of EDRF. In the defense against oxidative damage, there are enzymatic and nonenzymatic antioxidants. Regarding compounds used pharmacologically, we have been interested in ebselen, a seleno-organic compound exhibiting GSH peroxidase activity, which protects against reactive oxygen species generated, for example, at reoxygenation following a period of hypoxia. Further, we have studied lipoate and dihydrolipoate as antioxidant redox system and as singlet oxygen quencher, e.g. protecting against damage of deoxyguanosines in plasmid DNA generated by singlet oxygen.

Reactive oxygen species convey the cytotoxicity of several types of compounds (Fig. 1) [1,21, e.g. quinoid and nitroaromatic compounds and polyhalogenated alkanes such as carbon tetrachloride and halothane 133. Quinoid and nitroaromatic compounds may serve as redox cyclers, i.e. Correspondence to: H. Sies, Institut ftir Physiologische Chemie 1, Heinrich-Heine-Universitit Dusseldorf, Moorenstrasse 5, W-4000 Dusseldorf, Germany.

Sensitivity against reactive oxygen increased, e.g. due to antioxidant depletion

Fig. 1. Induction

of cell injury by reactive

oxygen species.

producing superoxide (OS?) at the expense of NADPH and 02 catalyzed by cellular reductases. Polyhalogenated alkanes are reductively metabolized to free radicals by microsomal cytochrome P450. The free radicals may attack polyunsaturated fatty acids of membrane phospholipids thus initiating lipid peroxidation. In this process the oxygen partial pressure plays a decisive role. The formation of the haloalkane free radicals at the heme moiety of cytochrome P-450 is inhibited by 02 since at that site 02 normally becomes activated during the monooxygenase cycle. On the other hand, 02 is necessary for the process of lipid peroxidation. Because of this dual and opposite role of 02, lipid peroxidation and thus cell injury, is preferentially induced at oxygen partial pressures between 1 and 10 mm Hg. An increased formation of reactive oxygen species may also occur upon resupply of 02 to a previously hypoxic tissue thus contributing to reoxygenation Oeperfusion) injury 141. In hepatocytes, reactive oxygen species are preferentially generated by the mitochondrial respiratory chain upon reoxygenation, but other enzymatic and non-enzymatic sources are also involved L51.Interestingly, release of reactive oxygen species does not cease when the cells lose their viability but continues for several hours. In this way non-viable cells may contnri injury of still viable cells in their vicinity (Fig. 1). Reactive oxygen species are also released by macrophages and granulocytes, specialized cell-types employed in defense (Fig. 1). Macrophages are activated by a variety of stimuli to release Odor nitric oxide (NO) 16-81. Activators of OTand thus Hz02 formation, due to spontaneous or enzyme-

catalysed dismutation of 02, include opsonized zymosan, phorbol esters and Ca2’ ionophores. In rat Kupffer cells Ogformation, which is catalyzed by a specific NADPH oxidase, may also be activated by exposure of the cells to hypoxia followed by reoxygenation [91. NO formation by catalysis of NO synthase is induced by a different set of stimuli including lipopolysaccharides and y-interferon. NO is derived from the guanidino nitrogen of L-arginine, and its synthesis is specifically blocked by the L-arginine analogue No-nitro-L-arginine [IO]. Both O?and NO alone already possess a significant cytotoxic potential. However, there may be a cooperative action in their damaging effects on target cells. OTand NO readily react yielding the peroxynitrite anion (ONOO-) [Ill. The latter decays rapidly once protonated to form the hydroxyl radical (OH) and nitrogen dioxide (NO&

O;+ NO' ____, ONOO- + H’ > ONOOH

-

ONOOOHOOH OH’ + NO2’

Oxidation of protein-SH in the presence of both Ogand NO and the induction of lipid peroxidation by ONOO- has been shown W&131. Likewise, the marked increase in chemiluminescence emitted by luminol in the presence of both 0~?02- and NO may also be due to the formation of the peroxynitrite anion and its subsequent decomposition 1141.In experiments with isolated rat Kupffer cells, phorbol 12-myristate 13-acetate-induced luminol chemiluminescence was doubled by the addition of L-arginine and significantly inhibited by NG-nitro+arginine, while the formation of Oiby NADPH oxidase was neither affected by L-arginine nor by NG-nitro-L-arginine. Beside its cytotoxic potential, NO possesses strong vasorelaxant properties and is thought to be the endothelium-derived relaxing factor (EDRF). Super-oxide dismutase may directly interact with NO catalyzing its conversion to the nitroxyl anion (NO3 [Xi], this being a novel function in addition to the “classical” dismutation of OTto Hz02 and Oz. NO- may be viewed as another form of EDRF. Reaction of superoxide dismutase with NO may be an additional explanation for the beneficial effects of the enzyme in tissue injuring processes where reactive oxygen species are considered to be involved. Superoxide dismutase targeted for endothelial cells diminished the elevated blood pressure in spontaneously hypertensive rats 1161. Similar to macrophages, granulocytes are activated by a variety of stimuli, including again opsonized zymosan, phorbol esters and Ca2’ iono-

phores, to release 02-/Hz02 In addition to O&I202 generated by NADPH oxidase, granulocytes may form HOC], catalysed by myeloperoxidase E171: HOC1 is a powerful oxidant and potential targets include amines, amino acids, thiols, thioethers, nucleotides and hemoproteins. Hz02 + Cl- + H’ ___,

H20 + HOC1

In the defence against oxidative damage, there are enzymatic and nonenzymatic antioxidants [181. Super-oxide dismutase, as an enzymatic antioxidant, has already been mentioned. Catalase and glutathione peroxidase catalyze the degradation of Hz02 to Hz0 and 02. The nonenzymatic antioxidants include lipophilic and hydrophilic compounds. a-Tocopherol and carotenoids are important lipid-soluble antioxidants while ascorbic acid (vitamin C) and glutathione belong to the water-soluble antioxidants. There are important interrelationships between these two systems. An example is the regeneration of a-tocopherol from the a-tocopheroxyl radical. a-Tocopherol (vitamin E, vit E-OH) rapidly reacts with peroxyl radicals (ROO) to generate the organic hydroperoxide (ROOH) and the a-tocopheroxyl radical kit E-O): ROO’ + vit E-OH

-

ROOH + vit E-O’

That way the radical function from the reactive organic radical (e.g., of a polyunsaturated fatty acid) is transposed into a less reactive chromanoxyl radical. The radical challenge is removed by reaction of the chromanoxyl radical (vit E-O) with a water-soluble hydrogen donor (AH), presumably ascorbic acid or glutathione: vit E-O’ + AH

_

vit E-OH + A’

Regarding antioxidants used pharmacologically, numerous compounds have been synthesized. We have been interested in ebselen, a selenoorganic compound exhibiting glutathione peroxidase activity [19,201. In a variety of biological systems, ebselen exhibited potent antioxidant properties and protected against reactive oxygen species generated, for example, at reoxygenation following a period of hypoxia. ACKNOWLEDGEMENT Supported by: Deutsche Forschungsgemeinschaft, Klinische Forschergruppe Leberschadigung, Str 92/4-3, and the National Foundation for Cancer Research, Bethesda.

551 REFERENCES

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Role of reactive oxygen species in cell toxicity.

Several types of compound exert their cytotoxicity by generating reactive oxygen species, notably the superoxide anion radical. These include quinoid ...
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