Impact of high glucose and transforming growth factor-ß on bioenergetic profiles in podocytes.

Diabetic nephropathy is the most common cause of chronic renal failure in industrialized countries. Depletion of podocytes plays an important role in the progression of diabetic glomerulopathy. Various factors in the diabetic milieu lead to serious podocyte stress driving the cells toward cell cycle arrest (p27(Kip1)), hypertrophy, detachment, and apoptosis. Mitochondria are responsible for oxidative phosphorylation and energy supply in podocytes. Recent studies indicated that mitochondrial dysfunction is a key factor in diabetic nephropathy. In the present study, we investigated metabolic profiles of podocytes under diabetic conditions. We examined oxygen consumption rates (OCRs) and oxidative phosphorylation complex activities in murine podocytes. Cells were exposed to high glucose for 48 hours, cultured for 10 passages under high-glucose conditions (30 mmol/L), or incubated with transforming growth factor-ß (5 ng/mL) for 24 hours. After prolonged exposure to high glucose, podocytes showed a significantly increased OCR at baseline and also a higher OCR after addition of oligomycin, indicating significant changes in mitochondrial energy metabolism. Higher OCRs after inhibition of respiration by rotenone also indicated changes in nonmitochondrial respiration. Podocytes stimulated with a proapoptotic concentration of transforming growth factor-ß displayed similar bioenergetic profiles, even with decreased citrate synthase activity. In all tested conditions, we found a higher cellular nicotinamide adenine dinucleotide content and changes in activities of respiratory chain complexes. In summary, we provide for the first time evidence that key factors of the diabetic milieu induce changes in glucose metabolism and mitochondrial function in podocytes.

Disruption of hepatic mitochondrial bioenergetics is not a primary mechanism for the toxicity of methoprene - relevance for toxicological assessment.

Methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate) is an insect growth regulator generally used to control insect populations by preventing insect maturation. So far, the effects of the insecticide on mitochondrial bioenergetics were not investigated. In the present work, liver mitochondria from Wistar rats were isolated and features of mitochondrial physiology were characterized in the presence of methoprene. High concentrations of methoprene, in the range of 40-100 nmol/mg of protein could decrease the transmembrane electric potential (Delta Psi) developed by mitochondria and, at the highest concentration, methoprene prevented complete Delta Psi repolarization after ADP addition. The effect was more evident using succinate than with ascorbate+TMPD as substrate. State 3 respiration was approximately 60% inhibited by 80 nmol of methoprene/mg of protein, while state 4 respiration, within the same range of methoprene concentrations, showed a slight increase, when both glutamate-malate and succinate were used as substrates. Additionally, FCCP-stimulated respiration was inhibited to an extent comparable to the effect on state 3, which suggests an interaction of methoprene with the respiratory chain, more evident with glutamate/malate as substrate. The activity of complex I (NADH-ubiquinone oxidorreductase) and that of the segment comprehending complexes II and III (succinate-cytochrome c reductase) were decreased in the presence of methoprene (approximately 60% and 85% of inhibition, respectively, with 300 nmol of methoprene/mg of protein), while the activities of cytochrome c oxidase and ATPase do not seem to be affected. Furthermore, the action of methoprene on the mitochondrial permeability transition was also studied, showing that the insecticide (in the range of 30-80 nmol mg(-1) of protein) decreases the susceptibility of liver mitochondria to the opening of the transition pore, even in non-energized mitochondria. These results lead to the conclusion that methoprene interference with hepatic mitochondrial function occurs only for high concentrations, which implies that the noxious effects of the insecticide reported for a number of non-target organisms are not fully attributable to mitochondrial effects. Therefore, it seems that mitochondrial activity does not represent the primary target for methoprene toxic action.

Evaluation of olive oil mill wastewater toxicity on the mitochondrial bioenergetics after treatment with Candida oleophila.

In a previous work the ability of Candida oleophila to use phenolic compounds as sole carbon and energy source at high concentrations without an additional carbon source was reported. C. oleophila grown in bioreactor batch cultures in a diluted and sterilized olive oil mill wastewater (OMW) caused a significant decrease in the total tannins content but no significant alteration was observed in phenolic acid and fatty acid content. Both treated and untreated OMWs were tested to evaluate the capacity in interfering with mitochondrial bioenergetics. Mitochondrial respiration was not affected by treated OMW on the range of used concentrations, contrary to the untreated OMW. Furthermore, mitochondrial membrane potential and respiratory complexes were always significantly less affected by treated OMW in comparison with untreated OMW. However, supplementary treatment should be applied before OMW could be considered non-toxic.

Target identification of drug induced mitochondrial toxicity using immunocapture based OXPHOS activity assays.

Mitochondrial dysfunction has been shown to be a pharmacotoxicological response to a variety of currently-marketed drugs. In order to reduce attrition due to mitochondrial toxicity, high throughput-applicable screens are needed for early stage drug discovery. We describe, here, a set of immunocapture based assays to identify compounds that directly inhibit four of the oxidative phosphorylation (OXPHOS) complexes: I, II, IV, and V. Intra- and inter-assay variation were determined and specificity tested by using classical mitochondrial inhibitors. Twenty drugs, some with known mitochondrial toxicity and others with no known mitochondrial liability, were studied. Direct inhibition of one or more of the OXPHOS complexes was identified for many of the drugs. Novel information was obtained for several drugs including ones with previously unknown effects on oxidative phosphorylation. A major advantage of the immunocapture approach is that it can be used throughout drug screening from early compound evaluation to clinical trials.

High-dose statins and skeletal muscle metabolism in humans: a randomized, controlled trial.

BACKGROUND: Myopathy, probably caused by 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibition in skeletal muscle, rarely occurs in patients taking statins. This study was designed to assess the effect of high-dose statin treatment on cholesterol and ubiquinone metabolism and mitochondrial function in human skeletal muscle. METHODS: Forty-eight patients with hypercholesterolemia (33 men and 15 women) were randomly assigned to receive 80 mg/d of simvastatin (n = 16), 40 mg/d of atorvastatin (n = 16), or placebo (n = 16) for 8 weeks. Plasma samples and muscle biopsy specimens were obtained at baseline and at the end of the follow-up. RESULTS: The ratio of plasma lathosterol to cholesterol, a marker of endogenous cholesterol synthesis, decreased significantly by 66% in both statin groups. Muscle campesterol concentrations increased from 21.1 +/- 7.1 nmol/g to 41.2 +/- 27.0 nmol/g in the simvastatin group and from 22.6 +/- 8.6 nmol/g to 40.0 +/- 18.7 nmol/g in the atorvastatin group (P = .005, repeated-measurements ANOVA). The muscle ubiquinone concentration was reduced significantly from 39.7 +/- 13.6 nmol/g to 26.4 +/- 7.9 nmol/g (P = .031, repeated-measurements ANOVA) in the simvastatin group, but no reduction was observed in the atorvastatin or placebo group. Respiratory chain enzyme activities were assessed in 6 patients taking simvastatin with markedly reduced muscle ubiquinone and in matched subjects selected from the atorvastatin (n = 6) and placebo (n = 6) groups. Respiratory chain enzyme and citrate synthase activities were reduced in the patients taking simvastatin. CONCLUSIONS: High-dose statin treatment leads to changes in the skeletal muscle sterol metabolism. Furthermore, aggressive statin treatment may affect mitochondrial volume.

Coenzyme q10 confers cardiovascular protection against acute mevinphos intoxication by ameliorating bioenergetic failure and hypoxia in the rostral ventrolateral medulla of the rat.

Coenzyme Q10 (CoQ10, ubiquinone) is a highly mobile electron carrier in the mitochondrial respiratory chain that also acts as an antioxidant. We evaluated the cardiovascular protective efficacy of CoQ10 at the rostral ventrolateral medulla (RVLM), a medullary site where sympathetic vasomotor tone originates and where the organophosphate poison mevinphos (Mev) acts to elicit cardiovascular intoxication. Experiments were carried out in adult male Sprague-Dawley rats that were maintained under propofol anesthesia. Microinjection bilaterally of Mev (10 nmol) into the RVLM induced progressive hypotension and minor bradycardia, alongside significant depression of the activity of NADH cytochrome c reductase (enzyme marker for Complexes I and III) or cytochrome c oxidase (enzyme marker for Complex IV) in the mitochondrial respiratory chain, reduction in ATP concentration, or tissue hypoxia in the RVLM. On the other hand, the activity of succinate cytochrome c reductase (enzyme marker for Complexes II and III) remained unaltered. The Mev-induced hypotension, bioenergetic failure, or hypoxia was significantly reversed when CoQ10 (4 microg) was coadministered bilaterally into the RVLM with the organophosphate poison. We conclude that CoQ10 confers cardiovascular protection against acute Mev intoxication by acting on the RVLM, whose neuronal activity is intimately related to the "life-and-death" process. We also showed that amelioration of the selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain, the reduced ATP level, and the induced tissue hypoxia in the RVLM are among some of the underlying mechanisms for the elicited protection.

Protection of mitochondrial system by Hippophae rhamnoides L. against radiation-induced oxidative damage in mice.

The whole extract of the fresh berries of Hippophae rhamnoides L. (RH-3), which has been reported to provide protection to whole mice, various tissues, cells and cell organelles against lethal irradiation, was further investigated for its effects on mitochondria isolated from mouse liver. Superoxide anion, reduced (GSH) and oxidized glutathione (GSSG) levels, NADH-ubiquinone oxidoreductase (complex I), NADH-cytochrome c oxidoreductase (complex I/II), succinate-cytochrome c oxidoreductase (complex II/III), mitochondrial membrane potential (MMP), lipid peroxidation (LPx) and protein oxidation (PO) were determined for RH-3-mediated radioprotective manifestation. Pre-irradiation treatment of mice with RH-3 (30 mg kg(-1,) i. p.; single dose; -30 min) significantly inhibited the radiation-induced increase in superoxide anions, GSSG, thiobarbituric acid reactive substances (TBARS), complex I, complex I/III activity and MMP maximally at 4 h (P < 0.05). This treatment inhibited the oxidation of proteins (P < 0.05) at all the time periods studied here. This study suggests that pre-irradiation treatment of mice with RH-3 protects the functional integrity of mitochondria from radiation-induced oxidative stress.

Coenzyme Q and the regulation of intracellular steady-state levels of superoxide in HL-60 cells.

The present work was set to study how CoQ concentrations affected steady-state levels of superoxide in a cellular model of partial CoQ(10) deficiency in cultured human myeloid leukemia HL-60 cells. Culturing HL-60 cells in the presence of p-aminobenzoate, a competitive inhibitor of polyprenyl-4-hydroxybenzoate transferase (Coq2p), produced a significant decrease of CoQ(10) levels without affecting cell viability. Concomitant decreases in CoQ-dependent electron transport activity and mitochondrial membrane potential were observed under these conditions. Intracellular superoxide was significantly elevated in cells treated with p-aminobenzoate, both under serum-containing and serum-free conditions, and this effect was reversed by exogenous CoQ(10). A slight increase of superoxide was also observed in CoQ(10)-supplemented cells in the absence of serum. Our results support a requirement for CoQ(10) to control superoxide levels in HL-60 cells. The importance of extramitochondrial sources of superoxide in cells with impaired CoQ(10) biosynthesis is discussed.

Modification of radiation damage to mitochondrial system in vivo by Podophyllum hexandrum: mechanistic aspects.

The present study was undertaken to investigate whether RP-1 treatment protected mitochondrial system against radiation damage and also to unravel the mechanism associated with this process. Radioprotection of mitochondrial system by Podophyllum hexandrum (RP-1) was investigated to understand its mechanism of action. Levels of superoxide anion (O2-), reduced or oxidized glutathione (GSH or GSSG), thiobarbituric acid reactive substance (TBARS), protein carbonyl (PC), ATP, NADH-ubiquinone oxidoreductase (complex-I), NADH-cytochrome c oxidoreductase (complex I/II), succinate-cytochrome c oxidoreductase (complex II/III) and mitochondrial membrane potential (MMP) were studied in mitochondria isolated from liver of mice belonging to various treatment groups. Whole body y-irradiation (10 Gy) significantly (p < 0.01) increased the formation of O2-, PC, and TBARS, upto 24 h as compared to untreated control. RP-1 treatment (200 mg/kg b.w.) to mice 2 h before irradiation reduced the radiation-induced O2- generation within 4 h and formation of TBARS and PC upto 24 h significantly (p < 0.01). Singularly irradiation or RP-1 treatment significantly (p < 0.01) increased the levels of glutathione within an hour, as compared to untreated control. Pre-irradiation administration of RP-1 enhanced levels of GSH induced increase in complex I (upto 16 h), complex I/III (4 h) complex II/III activity (upto 24 h; p < 0.01) and inhibited the radiation-induced decrease in MMP significantly (24 h; p < 0.01). The present study indicates that RP-1 itself modulates several mitichondrial perameters due to its influence on the biochemical milieu within and outside the cells. However, RP-1 treatment before irradiation modulates radiation induced perturbations such as the increase in electron transport chain enzyme activity, formation of O2-, TBARS and PC to offer radioprotection.

The herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) interacts with mitochondrial bioenergetic functions.

The effects of dicamba, a widely used broad-leaf herbicide, on rat liver mitochondrial bioenergetic activities were examined. The results obtained for state 4 respiration indicate not only an uncoupling effect, the result of an increase on the permeability of inner mitochondria membrane to protons, but also a strong inhibitory effect on the redox complexes. State 3 and respiration uncoupled by FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone) were inhibited to approximately the same extent, i.e. by about 70%. Depression of respiratory activity is essentially mediated through partial inhibition of mitochondrial complexes II and III. ATPase activity was much less depressed by dicamba than ATP synthase activity. Therefore, a considerable part of the inhibition observed on ATP synthase is the result of an inhibition on the redox complexes. The loss of phosphorylation capacity, induced by dicamba, was in the last analysis not only the result of a direct effect of dicamba on the enzymatic complex (F(0)-F(1) ATPase) but also the result of a deleterious effect on the integrity of the mitochondrial membrane, which can promote an inhibition of the respiratory complexes and an increase of the proton permeability of the inner membrane.

Effects of aging on mitochondrial enzyme activity of rat urinary bladder.

OBJECTIVES: Our previous study showed that aged rat bladders became fatigued faster than young bladders following repeated contraction induced by electrostimulation. One factor might be a lower energy-producing capability secondary to a decreased mitochondrial enzyme activity of the aged bladder. This study examined this possibility. MATERIALS AND METHODS: Mitochondria from 3- (n = 11) and 24-month-old (n = 10) Sprague-Dawley rats were isolated. Activities of the following enzymes were assayed: two key enzymes in the citric acid cycle, citrate synthase and malate dehydrogenase, and three enzymes in the respiratory chain reaction, NADH-cytochrome c reductase, succinate-cytochrome c reductase and cytochrome c oxidase. The concentration of phosphocreatine and ATP in the aged rat bladders and a separate group of young bladders (n = 12) was determined using high-performance liquid chromatography. RESULTS: (1) The aged bladders have a significantly lower level of phosphocreatine and ATP content than those of young bladders. (2) The activities of all five enzymes assayed were significantly lower in the aged bladders than in young bladders, especially for citrate synthase, which had only 46.8% of the activity of young bladders. CONCLUSIONS: Aging reduces the mitochondrial enzyme activity of the rat bladder resulting in a lower energy-production capability, which might explain some of the voiding dysfunctions found in the elderly.

Activities of mitochondrial oxidative phosphorylation enzymes in cultured amniocytes.

Amniocytes represent a population of foetal cells that can be used for prenatal diagnosis in families with suspected mitochondrial oxidative phosphorylation (OXPHOS) defects. In this paper, we present a complex protocol for evaluation of the function of mitochondrial OXPHOS enzymes in cultured amniocytes using three independent and complementary methods: (a) spectrophotometry as a tool for determination of the capacities of mitochondrial respiratory-chain enzymes (NADH ubiquinone oxidoreductase, succinate- and glycerophosphate cytochrome c reductase, cytochrome c oxidase and citrate synthase); (b) polarography as a tool for the evaluation of mitochondrial OXPHOS enzyme functions in situ using digitonin-permeabilised amniocytes (rotenone-sensitive oxidation of pyruvate+malate, antimycin A-sensitive oxidation of succinate, KCN-sensitive oxidation of cytochrome c, ADP-activated substrate oxidation) and (c) cytofluorometric determination of tetramethyl rhodamine methyl ester (TMRM) fluorescence in digitonin-permeabilised amniocytes as a sensitive way to determine the mitochondrial membrane potential under steady-state conditions (state 4 with succinate). These protocols are presented together with reference control values using 9-22 independent cultures of amniocytes.

A Ginkgo biloba extract (EGb 761) prevents mitochondrial aging by protecting against oxidative stress.

The effect of aging on indices of oxidative damage in rat mitochondria and the protective effect of the Ginkgo biloba extract EGb 761 was investigated. Mitochondrial DNA from brain and liver of old rats exhibited oxidative damage that is significantly higher than that from young rats. Mitochondrial glutathione is also more oxidized in old than in young rats. Peroxide formation in mitochondria from old animals was higher than in those from young ones. According to morphological parameters (size and complexity), there are two populations of mitochondria. One is composed of large, highly complex mitochondria, and the other population is smaller and less complex. Brain and liver from old animals had a higher proportion of the large, highly complex mitochondria than seen in organs from young animals. Treatment with the Ginkgo biloba extract EGb 761 partially prevented these morphological changes as well as the indices of oxidative damage observed in brain and liver mitochondria from old animals.

Antioxidative action of diterpenoids from Podocarpus nagi.

Diterpenoids, totarol (1), totaradiol (2), 19-hydroxytotarol (3), totaral (4), 4 beta-carboxy-19-nortotarol (5), sugiol (6), isolated from Podocarpus nagi, were evaluated as antioxidants. Microsomal lipid peroxidation induced by Fe(III)-ADP/NADPH and mitochondrial lipid peroxidation induced by Fe(III)-ADP/ NADH were inhibited by these terpenoids. They inhibited linoleic acid autoxidation but not generation of superoxide anion. Totarol (1) protected mitochondrial respiratory enzyme activities against NADPH induced oxidative injury. Totarane diterpenes from P. nagi were shown to be effective to protect biological systems and function against various oxidative stresses.

Effects of growth hormone in vitro on the glucose metabolism of fetal rat islet beta-cells.

This study was undertaken to investigate the effects of growth hormone (GH) on the in vitro maturation of the metabolism of fetal rat islets. For this purpose fetal islets were obtained from 21-day-old fetuses by mild collagenase digestion of the pancreas and cultured in RPMI 1640 supplemented with 10% fetal calf serum. After one day the medium was changed and supplemented with 1% fetal calf serum with or without GH (1 microgram/ml, human recombinant) and the islets cultured for another two days. Islets were then studied with regard to insulin secretion, (pro)insulin and total protein biosynthesis, glucose utilization and oxidation, thymidine incorporation, insulin and DNA contents and the contents of mRNAs for either insulin, adenine nucleotide translocator or cytochrome b. In addition, the activities of glucose phosphorylating enzymes and succinate-cytochrome c reductase were measured. Islets treated with GH showed increased insulin secretion in response to glucose, increased rates of glucose oxidation and utilization, increased thymidine incorporation and increased activities of succinate cytochrome c reductase and glucose phosphorylation at high glucose concentrations. There were, however, no changes in (pro)insulin and total protein biosynthesis, contents of insulin and DNA or the contents of any of the mRNAs. These combined data show that fetal beta-cells are sensitive to growth hormone with respect to glucose metabolism, insulin release and DNA replication. The increased rates of islet glucose phosphorylation may reflect glucokinase activity and explain part of the increased insulin responsiveness to glucose of the fetal rat beta-cell. These observations suggest that GH is of physiological significance for the maturation of the fetal beta-cell.

Riboflavin-responsive complex I deficiency.

Three patients from a large consanguineous family, and one unrelated patient had exercise intolerance since early childhood and improved by supplementation with a high dosage of riboflavin. This was confirmed by higher endurance power in exercise testing. Riboflavin had been given because complex I, which contains riboflavin in FMN, one of its prosthetic groups, had a very low activity in muscle. Histochemistry showed an increase of subsarcolemmal mitochondria. The low complex I activity contrasted with an increase of the activities of succinate dehydrogenase, succinate-cytochrome c oxidoreductase and cytochrome c oxidase. Isolated mitochondria from these muscle specimens proved deficient in oxidizing pyruvate plus malate and other NAD(+)-linked substrates, but oxidized succinate and ascorbate at equal or higher levels than controls. Two years later a second biopsy was taken in one of the patients, and the activity of complex I had increased from 16% to 47% of the average activity in controls. In the four biopsies, cytochrome c oxidase activity correlated negatively with age. We suspect that this is due to reactive oxygen species generated by the proliferating mitochondria and peroxidizing unsaturated fatty acids of cardiolipin. Three of the four patients had low blood carnitine, and all were found to have hypocarnitinemic family members.

Inhibition of brain macrophage/microglial respiratory chain enzyme activity in experimental autoimmune encephalomyelitis of the Lewis rat.

We measured the activities of five respiratory chain enzymes in brain macrophages/microglial cells from Lewis rats with experimental autoimmune encephalomyelitis (EAE) and found a significant reduction of the activity of nicotinamide adenine dinucleotide-dehydrogenase (NADH-DH), succinate cytochrome c reductase (SCCR) and succinate dehydrogenase (SDH) when compared with age-matched healthy control animals. The inhibition of NADH-DH (complex I) was specific for EAE, while we also found a reduction of SCCR and SDH activities (complex II) in newborn rats and adjuvant-immunised rats. Activities of NADH cytochrome c reductase (NCCR) and cytochrome c oxidase (COX) were not significantly changed. These observations demonstrate an impairment of brain macrophage/microglial respiratory chain function in central nervous system inflammation.

AZT causes tissue-specific inhibition of mitochondrial bioenergetic function.

The mitochondrial myopathy associated with long-term AZT therapy is a factor that limits the clinical efficacy of this compound in the treatment of AIDS. The biochemical basis for this tissue-specific pathology was investigated by measuring the effect of AZT on various aspects of bioenergetic function in mitochondria isolated from rat skeletal muscle, brain, and liver. AZT induced a dose-dependent inhibition of both NADH-linked respiration in intact mitochondria and NADH-cytochrome c reductase activity (but not succinate-cytochrome c reductase activity) in freeze-thawed mitochondrial preparations isolated from all three tissue types (1/2 maximal inhibition was obtained at 2 mg/ml and between 0.3 and 0.8 mg/ml AZT, respectively). These data demonstrate that high concentrations of AZT inhibit electron transfer through respiratory enzyme complex I. Moreover, AZT was shown to induce a tissue-specific inhibition of succinate-linked respiration in intact mitochondria isolated from rat skeletal muscle (1/2 maximal inhibition at 0.5 mg/ml AZT) and possibly brain, but not liver. The data suggest that this inhibition possibly occurs at the level of succinate transport. These results may help to explain the tissue-specific mitochondrial effects that are induced by long-term zidovudine treatment of AIDS patients and suggest that the anti-retroviral activity exhibited by AZT may be distinct from its mechanism of mitochondrial toxicity.

Mitochondrial toxicity of cationic photosensitizers for photochemotherapy.

The triarylmethane derivative Victoria Blue-BO (VB-BO) and the chalcogenapyrylium (CP) dyes have potential for use in photochemotherapy, because they are taken up by the mitochondria of malignant cells and cause cell death. To clarify the mechanism of cell killing we examined the phototoxic effects of VB-BO and a series of three CP dyes on bioenergetic function in isolated rat liver mitochondria. Without photoirradiation, and irrespective of the respiratory substrate used, each of the compounds tested induced some uncoupling of oxidative phosphorylation. Visible irradiation of VB-BO produced an inhibition of mitochondrial respiration when glutamate plus malate, but not succinate, was used as the respiratory substrate. With photoirradiation VB-BO was also shown to inhibit rotenone-sensitive NADH-cytochrome c reductase activity, but it had no effect on succinate-cytochrome c reductase activity. These data indicate that photoactivation of VB-BO produces selective inhibition of mitochondrial respiratory complex I. Photoirradiation of the CP dyes inhibited both complex I and complex II initiated respiratory activity. With photoirradiation, the CP dyes also inhibited both NADH- and succinate-cytochrome c reductase activities, as well as other membrane-bound enzymes, cytochrome c oxidase and succinate dehydrogenase, but not the mitochondrial matrix enzyme, citrate synthetase, or the cytosolic enzyme, lactate dehydrogenase. alpha-Tocopherol protected bioenergetic activities against CP dye photodamage. These results suggest that mitochondrial photosensitization by CP compounds is mediated by the production of membrane-damaging singlet oxygen which causes nonspecific damage to membranes and membrane-bound enzymes.

Differential modulation of rat heart mitochondrial membrane-associated enzymes by dietary lipid.

Diets supplemented with high levels of saturated fatty acids derived from sheep kidney (perirenal) fat or unsaturated fatty acids derived from sunflower seed oil were fed to rats and the effect on heart mitochondrial lipid composition and membrane-associated enzyme behaviour was determined. The dietary lipid treatments did not change the overall level of membrane lipid unsaturation but did alter the proportion of various unsaturated fatty acids. This led to a change in the omega 6/omega 3 unsaturated fatty acid ratio, which was highest in the sunflower seed oil fed rats. Arrhenius plots of the mitochondrial membrane associated enzymes succinate-cytochrome c reductase and oligomycin-sensitive adenosinetriphosphatase (ATPase) after dietary lipid treatment revealed different responses in their critical temperature. For succinate-cytochrome c reductase, the critical temperature was 29 degrees C for rats fed the sheep kidney fat diet and 20 degrees C for rats fed the sunflower seed oil diet. In contrast, no shift in the critical temperature for the mitochondrial ATPase was apparent as a result of the differing dietary lipid treatments. The results suggest that the discontinuity in the Arrhenius plot of succinate-cytochrome c reductase is induced by some change in the physical properties of the membrane lipids. In contrast, mitochondrial ATPase appears insensitive, in terms of its thermal behaviour, to changes occurring in the composition of the membrane lipids. However, the specific activity of the mitochondrial ATPase was affected by the dietary lipid treatment being highest for the rats fed the sheep kidney fat diet. No dietary lipid effect was observed for the specific activity of succinate-cytochrome c reductase. This differential response of the two mitochondrial membrane enzymes to dietary-induced changes in membrane lipid composition may affect mitochondrial oxidative phosphorylation.