Sequential damage in mitochondrial complexes by peroxidative stress

Neurochemical Research, Vol. 16, No. 12, 1991, pp. 1295-1302

Sequential Damage in Mitochondrial Complexes By Peroxidative Stress G. Benzi 1, D. Curti 1, O. Pastoris 1, F. Marzatico 1, R. F. Villa x, and F. Dagani I (Accepted June 12, 1991)

The biochemical characteristics of the electron transfer chain are evaluated in purified non-synaptic ("free") mitochondria from the forebrain of 60-week-old rats weekly subjected to peroxidative stress (once, twice, or three times) by the electrophilic prooxidant 2-cyclohexene-l-one. The following parameters are evaluated: (a) content of respiratory components, namely ubiquinone, cytochrome b, cytochrome cl, cytochrome c, (b) specific activity of enzymes, namely citrate synthase, succinate dehydrogenase, rotenone-sensitive NADH" cytochrome c reductase, cytochrome oxidase; (c) concentration of reduced glutathione (GSH). Before the first peroxidative stress induction, the rats are administered for 8 weeks by intraperitoneal injection of vehicle, papaverine, ~-yohimbine, almitrine or hopanthenate. The rats are treated also during the week(s) before the second or third peroxidative stress. The cerebral peroxidative stress induces: (a) initially, a decrease in brain GSH concentration concomitant with a decrease in the mitochondrial activity of cytochrome oxidase of aa3-type (complex IV), without changes in ubiquinone and cytochrome b populations; (b) subsequently, an alteration in the transfer molecule cytochrome c and, finally, in rotenone-sensitive NADH-cytochrome c reductase (complex I) and succinate dehydrogenase (complex II). The selective sensitivity of the chain components to peroxidative stress is supported by the effects of the concomitant subchrouic treatment with agents acting at different biochemical steps. In fact, almitrine sets limits to its effects at cytochrome c content and aa3-type cytochrome oxidase activity, while 8-yohimbine sets limits to its effects at the level of tricarboxylic acid cycle (citrate synthase) and/or of intermediary between tricarboxylic acid cycle and complex II (succinate dehydrogenase). The effects induced by sequential peroxidative stress and drug treatment are supportive of the hypothesis that leakage of electrons (as a mandatory side-effect of the normal flux of electrons from both NADH and succinate to molecular oxygen) would be due to alteration in both availability of GSH and the content of components in the respiratory chain associated to energy-transducing system. In this field there is a cascade of derangements involving, at the beginning, the complex IV and, subsequently, other chain components, including cytochrome c and, finally, complexes II and I. KEY WORDS: Electron transfer chain damage; sequential peroxidative stress; ~-yohimbine; almitrine; papaverine; hopanthenate.

INTRODUC~ON

human and non-human primates (1,2) because of its accumulation by the dopaminergic re-uptake system (3) supporting the degeneration of neurons in the nigrostriatal structure, as occurs in Parkinson's disease (4,5). It has been proposed that MPP + inhibits complex I of the mitochondrial electron transfer chain causing cell death via a derangement of the aerobic energy metabolism. In L-dopa-treated patients who died with Parkin-

The MAO B-catalyzed conversion of MPTP (1-methyl4-phenyl-l,2,3,6-tetrahydropyridine) to MPP + (1-methyl4-phenyl-pyridinium) causes Parkinson-like symptoms in 1 Institute of Pharmacology, Faculty of Sciences, University of Pavia, Piazza Botta 11, 27100 Pavia, Italy.

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Benzi, Curti, Pastoris, Marzatico, Villa, and Dagani

son's disease, rotenone-sensitive NADH:cytochrome c reductase activity (related to complexes I and III) seems to be decreased, while antimycin A-sensitive succinate:cytochrome c reductase activity (related to complexes II and III) seems to be normal, supporting the hypothesis of an impairment of the complex I (6-10). The complex I-inhibitor rotenone binds to one of the seven subunits of complex I encoded by mitochondrial DNA (11). A moderate decrease in three subunits of complex I in striatum from Parkinson's disease patients was described (8). Furthermore, by the polymerase chain reaction (PCR) technique, small amounts of the mitochondrial DNA deleted genome in the striatum of patients with Parkinson's disease and senescence were detected (12). In contrast, by the Southern blot analysis no alteration of the mitochondrial DNA was detectable in the substantia nigra and other brain regions of patients with Parkinson's disease (13). The repetition of the same experiment using the PCR technique showed that in addition to the numerous bands, the deleted genome was present even in control aged subjects. This result suggests that the deleted genome is not a specific property found in patients with Parkinson's disease, but rather a result of aging per se (14). Thus, the reduction in enzyme activity of the complex I may be also due to alteration of nuclear encoded subunits, or modification of the enzyme assembly process, or direct damage by peroxidative stress generating oxygen free-radicals. In accordance with the experiments performed in human subjects, to test the influence of peroxidative stress, the characteristics of the electron transfer chain should be evaluated in non-synaptic ("free") mitochondrial fraction including both neuronal and glial mitochondria. In this research, the fraction is obtained from the forebrain of rats weekly subjected to peroxidative stress by the electrophilic prooxidant 2-cyclohexene-l-one producing a depletion of cerebral reduced glutathione (GSH). In fact, GSH depletion may be produced by diazene derivatives oxidizing GSH, sulphydryl reactants alkylating GSI-I, compounds inhibiting GSH synthesis, and agents bearing an electrophilic site. Particularly, diethyl maleate and cycIohexene-l-one decrease cerebral GSH due to their conjugation with GSH, catalyzed by glutathione S-transferases. Diethyl maleate reveals the strongest actMty; it is, however, rapidly hydrolyzed by esterase to generate maleate which produces many other biochemical effects. Thus, the prooxidant cyclohexene-1one seems to be more suitable as GSH depletor (15-17). Furthermore, to test which are the factors and how they are involved in the peroxidative stress, the influence of some agents acting at different biochemical levels

may be useful. In general, the major factors involved are represented by cerebral circulation, carbohydrate metabolism, oxygen availability, and/or catecholaminergic activity. Thus, the biochemical evaluations are performed also after the subchronic pretreatment with agents acting on cerebral macrocirculation (i.e., papaverine), carbohydrate metabolism (i.e., hopanthenate), oxygen partial pressure (i.e., almitrine), and catecholaminergic system (i.e., ~-yohimbine).

EXPERIMENTAL PROCEDURE Animals and Experimental Plan. Male albino Wistar rats aged 60 weeks are used. The animals are kept under constant environmental conditions (temperature: 22 ___ I~ relative humidity: 60 -+ 5%; circadian rhythm: 12 hr light and 12 hr dark) and fed normal laboratory diet as pellets. As shown in Figure 1, lots of 5 rats are weekly subjected to peroxidative stress, once, twice or three times. The prooxidant 2cyclohexene-l-one is used at the dose able to incluce a severe reversible depletion of cerebral GSH = 100 mg/kg, i.p. (15-17). Before the first peroxidative stress induction, the rats are administered for 8 weeks (7 days a week) by intraperitoneal injection of: (a) vehicle (0.5% methylcellulose saline); (b) papaverine = 1 mg/kg; (c) 8-yohimbine (raubasine) = 1 mg/kg; (d) almitrine = 3 mg/kg; (e) hopanthenate = 100 mg/kg. The rats are treated also during the week(s) before the second or third peroxidative stress. The doses are within the range used experimentally to characterize the pharmacodynamic action of the different substances tested. The allocation of the rats to the different lots are made randomly. The forebrains are analyzed 18 hours after the induction of the peroxidative stress. Analytical Techniques. At the scheduled time the animals are sacrificed by decapitation and their forebrains are quickly dissected out (18). Briefly, the brain is collected within 20-30 sec after the rat decapitation in ice-cold isolation medium (IM = 320 mM sucrose, 1

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Mitochondrial Damage by Sequential Peroxidative Stress mM EDTA, 10 mM Tris-HC1, pH 7.4). The tissue is minced in small pieces, rinsed several times with IM and homogenized in the same medium (1 g/10 ml) with a Dounce-type glass-glass homogenizer by 12 strokes up-and.down. The homogenate is centrifuged as described (18) without modification except that the Ficoll-sucrose density gradient ultracentrifugation is carried out with a rotor SW 28.1 spinning at 99,000 gay for 30 min in Beckman L5-50 ultracentrifuge (19). The resultingpellet of non-synaptic("free") mitochondriais washed with IM containing bovine serum albumin (0.5 mg/ml). The final pellet is suspended in IM at the protein concentration of 6-9 mg/ml. The following parameters are evaluated: (a) concentration (nmol/mg protein) of respiratory components: ubiquinone, cytochrome b, cytochrome cl, cytochrome c (20-23); (b) specific activity (nmol/mirgmgprotein) of enzymes: citrate synthase, suceinate dehydrogenase, rotenone-sensitive NADH: cytochrome e reductase, cytochrome e oxidase (18,24). To evaluate the GSH concentration, at the scheduled time the forebrains are immediately powdered under liquid nitrogen and stored at - 80~ The assay in frozen forebrain powder is carried out within 3 hours (25-28). Briefly, the frozen brain powder is layered into 200300 ILl of 3 M perchloric acid with mixing, and diluted with ice-cold 5 mM EDTA. The suspension is centrifuged at 6,000 g for 45 min at 4~ and the supernatant fluid is neutralized with 2 M potassium bicarbonate and centrifuged. An aliquot of neutralized perchlorate extract (about 3-6 ~1) is analyzed for total glutathione (28). The reaction mixture contains 0.4 mM DTNB, 0.17 mM NADPH, 16 ~g glutathione reductase, 5 mM EDTA and 100 mM potassium phosphate buffer, pH 7.2. The rate of reduction of DTNB is continuously monitored at 412 nm (25). The concentration of oxidized glutathione is obtained utilizing an aliquot (50-100 ILl) of the neutralized perchlorate extract added by 2-vinylpyridine as an -SH trap (27). After incubating at 20~ for 60 min, the neutralized mixture is assayed by the DTNB method (25,28). StatisticalAnalysis. Two statistical tests (ANOVA and Dunnett's tests) are applied to the results (p