Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation.

K Schulze-Osthoff,A.C Bakker,B Vanhaesebroeck,R Beyaert,W.A Jacob,W Fiers

doi:10.1016/s0021-9258(18)42768-8

K Schulze-Osthoff, A.C Bakker + Show 4 more

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https://doi.org/10.1016/s0021-9258(18)42768-8

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Journal: The Journal of biological chemistry	Publication Date: Mar 1, 1992
Citations: 1034	License type: cc-by

Abstract

Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor necrosis factor (TNF). Using various inhibitors of mitochondrial electron transport we have investigated the mechanism of TNF-mediated cytotoxicity in L929 and WEHI 164 clone 13 mouse fibrosarcoma cells. Inhibitors with different sites of action modulated TNF cytotoxicity, however, with contrasting effects on final cell viability. Inhibition of mitochondrial electron transport at complex III (cytochrome c reductase) by antimycin A resulted in a marked potentiation of TNF-mediated injury. In contrast, when the electron flow to ubiquinone was blocked, either at complex I (NADH-ubiquinone oxidoreductase) with amytal or at complex II (succinate-ubiquinone reductase) with thenoyltrifluoroacetone, cells were markedly protected against TNF cytotoxicity. Neither uncouplers nor inhibitors of oxidative phosphorylation nor complex IV (cytochrome c oxidase) inhibitors significantly interfered with TNF-mediated effects, ruling out the involvement of energy-coupled phenomena. In addition, the toxic effects of TNF were counteracted by the addition of antioxidants and iron chelators. Furthermore, we analyzed the direct effect of TNF on mitochondrial morphology and functions. Treatment of L929 cells with TNF led to an early degeneration of the mitochondrial ultrastructure without any pronounced damage of other cellular organelles. Analysis of the mitochondrial electron flow revealed that TNF treatment led to a rapid inhibition of the mitochondria to oxidize succinate and NADH-linked substrates. The inhibition of electron transport was dose-dependent and became readily detectable 60 min after the start of TNF treatment, thus preceding the onset of cell death by at least 3-6 h. In contrast, only minor effects were observed on complex IV activity. The different effects observed with the mitochondrial respiratory chain inhibitors provide suggestive evidence that mitochondrial production of oxygen radicals mainly generated at the ubisemiquinone site is a causal mechanism of TNF cytotoxicity. This conclusion is further supported by the protective effect of antioxidants as well as the selective pattern of damage of mitochondrial chain components and characteristic alterations of the mitochondrial ultrastructure.

Highlights

Inhibition of mitochondrial electron transport at complex I11 by antimycin A resulted in a marked potentiatoiofnTNF- Tumor necrosis factor (TNF),’ primarily produced by actimediated injury
Several cellular pathways have been suggested leading to cell death, including perturbation of ion homeostasis, activation of proteases and phospholipases, generation of reactive oxygen specieso, r degradation of DNA[31].Despite a growing number of investigations dealing mainly with immunomodulatory activities of TNF, itsmechanism of cytotoxicity is still largely unknown
The necrotic form of cell death is mainly characterized by swelling of the cytoplasm and organelles followedby lysis of the plasma membrane

Summary

MATERIALS AND METHODS

Bation times ranging from 1to 17 h, cells were processed for electron microscopy. The growth medium was removed by two changes with. (Deisenhofen, Federal Republic of Germany): actinomycin D, adenosine diphosphate, amytal (amobarbital), butylated hydroxyanisole, butylated hydroxytoluene, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone the mitochondrial respiratory chain is shown in Fig. 1.Inhibitors of complex 111, which inhibit the electron flow from ubiquinone to cytochrome cl, strongly potentiated TNF-me-. Amytal strongly decreased TNF susceptibility (Table I; Fig.2b),and rotenone, which inhibits the same step, was protective, albeit to a lesser extent (TableI).when the electron transport from succinate dehydrogenase wasblockedby the addition of 24 h of further incubation, cell viability was routinely determined via TTFA, TNF cytotoxicity was reduced (Table I). Iron chelators, andantioxidants on TNF cytotoxicity in L929 cells in the presence and absence of actinomycin D (Act D). Data obtained with drug concentrations resulting in cell survival higher than 65% in the absence of TNF are shown.SOD, superoxide dismutase.

11. Uncouplers

Findings

DISCUSSION