High-amplitude Swelling Of Mitochondria Research Articles

Holographic imaging of the mitochondrial swelling in the living cells reveals that mitochondrial depolarization occurs before activation of the high-conductance permeability transition pore

Mitochondrial permeability transition is caused by opening of the Cyclosporine A (CSA) dependent calcium-induced pore, Permeability Transition Pore (PTP). PTP activation is the central event in stress-induced cell death. Molecular details of PTP opening remain incompletely understood. PTP makes mitochondrial inner membrane permeable to the molecules up to 1.5 kDa in size and results in high-amplitude mitochondrial swelling. Swelling and solutes equilibration make mitochondria optically transparent—the phenomenon that has been widely used as a bona fide “light-scattering” PTP assay in the isolated mitochondria.

Mechanism of triclosan toxicity: Mitochondrial dysfunction including complex II inhibition, superoxide release and uncoupling of oxidative phosphorylation

Triclosan (5-chloro-2′-(2,4-dichlorophenoxy)phenol), a widely used antibacterial agent, exerts adverse effects on the organism of mammals. Recent research reviled that triclosan at low micromolar concentrations causes mitochondrial dysfunction in many cell types, but the mechanisms of its effect are not fully understood. Here we show that exposure to triclosan disrupted membrane potential, prevented the calcium uptake-driven high-amplitude mitochondrial swelling, stimulated the respiration in the presence of complex I substrates, and suppressed the ADP-stimulated respiration in the presence of complex II substrate, succinate. Triclosan directly inhibited complex II activity. Similar to the complex II inhibitor thenoyltrifluoroacetone, triclosan induced the oxidation of the cytochromes b566 and b562 and caused the release of mitochondrial superoxide. Opposite to thenoyltrifluoroacetone, triclosan increased superoxide release synergistically with myxothiazol but not with antimycin A, indicating different topology of superoxide-producing sites. We concluded that triclosan is unique by its capability of acting as both a protonophore and an unusual complex II inhibitor, which interferes with the mitochondrial respiration by blocking the electron transfer between ubiquinone at the Qd-binding site and heme b. Our data can provide an insight into the mechanisms of the carcinogenic effect of triclosan in the liver and other tissues.

On the “swelling” of mitochondria under palmitic acid, calcium, and hypotension treatment

The high-amplitude swelling of mitochondria is critically considered. In contrast to numerous statements by some authors about a marked swelling of isolated liver mitochondria under the influence of palmitic acid, calcium ions, or hypotension, we have shown that mitochondria are generally not subject to highamplitude swelling. According to optical-microscopy data even during long-lasting incubation (in distilled water) where full hypotension takes place, the size of liver mitochondria (approximately 1 µm) can be enlarged by no more than by 40%. Under short-lasting hypotension or the addition of palmitic acid the mitochondrial diameter becomes greater by only 20% or remains virtually unchanged. The light scattering of the mitochondrial suspension measured using a photometer according to the decrease in optical density declines by 2.5 times. A decrease in the light scattering in hypotension or via the addition of palmitic acid or calcium (in an isotonic medium) occurs because of damage (even destruction) to the outer membrane, rather than due to the swelling of mitochondria, as was previously believed. The inner membrane is not significantly expanded. The destruction of the outer membrane reduces the probability of light scattering by each mitochondrion at the boundary layer of the water/membrane interface. Release of substances from the matrix resulting in a decrease of its refractive index may additionally contribute to the decrease in light scattering. Palmitic acid and calcium (at concentrations of 10 to 100 µM) cause permeabilization and disruption of the outer membrane gradually, over several minutes. Full hypotension activates this process very rapidly, viz., within a fraction of a second. Under low ionic-strength conditions, the addition of calcium leads to neutralization of negative charges on the membrane surface, which induces aggregation of mitochondria, thus enhancing light scattering and creating the illusion of mitochondrial swelling.

The effect of modulators of large-conductance Ca2+-modulated K+ channels on rat AS-30D ascites hepatoma cells and isolated liver mitochondria treated with Cd2+

ATP-producing cell organelles, mitochondria, are a primary target of heavy metals, major environmental pollutants causing a variety of diseases and pathologies. The mechanism of heavy metal toxic effect was established to include changes both in the intracellular production of reactive oxygen species (ROS) and mitochondrial dysfunction due to respiratory chain disorders and activation of the Ca2+-dependent non-selective pore in the mitochondrial inner membrane. Te role of other ion channels including such selective potassium channels as large-conductance Ca2+-activated K+ channels, BK(Ca), supposed to be cytoprotective, remains poorly studied. In the present work conducted on rat AS-30D ascites hepatoma cells and liver mitochondria, we studied the effect of different BK(Ca) effectors in the presence or absence of Cd2+ ions in the incubation medium, specifically of two activators, NS1619 and NS004, and one blocker, paxilline. After 24-h incubation of AS-30D cells with 10 μM of both NS1619 and NS004, the number of apoptized cells was found to increase significantly versus control; moreover, the presence of these BK(Ca) activators in the incubation medium exerted an additive effect on Cd2+-induced apoptosis of AS-30D cells. The same concentration of NS1619 and NS004 did not affect significantly AS-30D cellular respiration after 3, 24 and 48 h of incubation, although increasing after 3 h the intracellular production of ROS. In experiments on isolated rat liver mitochondria, NS1619 and NS004 added at the same concentration to the KCl-containing medium did not affect the rates of respiratory State 3 (after Chance) and maximally uncoupled respiration (both in the presence and absence of Cd2+); concurrently, they induced a weak uncoupling effect by increasing both basal and resting-state (State 3 after Chance) respirations, and also enhanced a high-amplitude mitochondrial swelling induced by Cd2+ in this medium. Paxilline (at 1μM) was shown to reduce the mortality of AS-30D cells after 3-, 24- and 48 h-incubation in the presence of Cd2+ and to increase the intracellular ROS production in control after 3 and 24 h of exposure. Paxilline added at a concentration exerting a long-term protective effect did not affect cellular respiration of rat AS-30D cells and isolated liver mitochondria (both in the presence and absence of Cd2+) and did not reduce mitochondrial swelling observed in the presence of Cd2+ and BK(Ca) activators. Possible molecular action mechanisms of BK(Ca) modulators are discussed.

Induction of Ca2+-dependent cyclosporin A-insensitive nonspecific permeability of the inner membrane of liver mitochondria and cytochrome c release by α,ω-hexadecanedioic acid in media of varying ionic strength.

In liver mitochondria loaded with Ca2+ or Sr(2+), α,ω-hexadecanedioic acid (HDA) can induce nonspecific permeability of the inner membrane (mitochondrial pore) by the mechanism insensitive to cyclosporin A (CsA). In this work we studied the effect of ionic strength of the incubation medium on the kinetics of the processes that accompany Ca2+-dependent induction of the mitochondrial pore by fatty acid: organelle swelling, Ca2+ release from the matrix, changes in transmembrane potential (Δψ) and rate of oxygen consumption, and the release of cytochrome c from the intermembrane space. Two basic incubation media were used: sucrose medium and isotonic ionic medium containing KCl without sucrose. We found that 200 μM Ca2+ and 20 μM HDA in the presence of CsA effectively induce high-amplitude swelling of mitochondria both in the case of sucrose and in the ionic incubation medium. In the presence of CsA, mitochondria can rapidly absorb Ca2+ and retain it in the matrix for a while without reducing Δψ. Upon incubation in the ionic medium, mitochondria retain most of the added Ca2+ in the matrix for a short time without reducing the Δψ. In both cases the addition of HDA to the mitochondria 2 min after the introduction of Ca2+ leads to the rapid release of these ions from the matrix and total drop in Δψ. The mitochondrial swelling induced by Ca2+ and HDA in non-ionic medium is accompanied by almost maximal stimulation of respiration. Under the same conditions, but during incubation of mitochondria in the ionic medium, it is necessary to add cytochrome c for significant stimulation of respiration. The mitochondrial swelling induced by Ca2+ and HDA leads to the release of cytochrome c in a larger amount in the case of ionic medium than for the sucrose medium. We conclude that high ionic strength of the incubation medium determines the massive release of cytochrome c from mitochondria and liberates it from the respiratory chain, which leads to blockade of electron transport along the respiratory chain and consequently to disruption of the energy functions of the organelles.

Physiological modulators of cyclosporin A-insensitive nonspecific permeability of the inner membrane of liver mitochondria induced by calcium ions and α,ω-hexadecanedioic acid

ity, inhibited mitochondrial swelling induced by Ca 2+ and HDA, despite the presence of succinate in the incubation medium. Natural uncouplers of oxidative phosphorylation, oleic and linoleic acids, produced a similar effect. These data suggest that energization of organelles, which promotes Ca 2+ transport into the matrix, is one of the basic requirements of pore opening in liver mitochondria induced by Ca 2+ and HDA. It is shown that ATP at the physiological concentration of 2 mM inhibits HDAinduced highamplitude swell� ing of mitochondria by reducing free Ca 2+ concentration in the medium. ADP at the same concentration had a similar effect. This modulating effect of nucleotides apparently is attributable to their ability to chelate cal� cium ions. Polycation spermine, which is known as an inhibitor of the classical CsAsensitive pore, at the physiological concentration of 1 mM inhibited CsAi nsensitive swelling of liver mitochondria induced by sequential addition of Ca 2+ and HDA. It is assumed that such action of spermine is due to its ability to shield the negative surface charges on the inner membrane of mitochondria. Bovine serum albumin (BSA), which is able to bind free fatty acids and thus prevent the induction of Ca 2+ �dependent pore, inhibited HDA� induced swelling of mitochondria. However, at the same BSA/fatty acid molar ratio inhibitory effect of BSA was much less pronounced if HDA was used as the pore inducer instead of palmitic acid. Apparently, this can be accounted by the fact that BSA binds α,ω�dioic acids weaker than their monocarboxylic analogues.

Spermine Selectively Inhibits High-Conductance but not Low-Conductance Mode of the Mitochondrial Permeability Transition Pore (MPTP)

Spermine is a biological organic polymer that has four primary amino groups. It is involved in the regulation of transcription, cell signalling and enzymatic activity. It has also been demonstrated that polyamines inhibit calcium-induced high amplitude mitochondrial swelling, suggesting their involvement in regulation of the mitochondrial Permeability Transition Pore (mPTP). Here we further investigated the mechanisms of polyamine inhibition of calcium-induced, cyclosporine A (CSA) sensitive mPTP.Experiments were performed using isolated rat liver mitochondria resuspended in sucrose based recording solution. mPTP was induced by the addition of calcium to energized mitochondria. mPTP activation was measured by three independent approaches: 1) calcium release from the mitochondria detected with calcium green; 2) mitochondrial membrane depolarization using TMRM probe and 3) mitochondrial swelling measured as a decrease of light absorbance. In control experiments, in the presence of the specific inhibitor CSA, mPTP inhibition was observed by all three approaches. However, in the presence of spermine (0.02 mM to 0.2 mM) only mitochondrial swelling was inhibited, while membrane depolarization and calcium release were not affected.These results suggest that spermine inhibited high-conductance mode of mPTP, which opening is required for induction of mitochondrial swelling in sucrose based media. However spermine did not inhibit low-conductance mode mPTP. Our data are consistent with possible distinct regulation and/or nature of the different modes of mPTP.

Accumulation of dodecyltriphenylphosphonium in mitochondria induces their swelling and ROS-dependent growth inhibition in yeast

Hydrophobic cations with delocalized charge are used to deliver drugs to mitochondria. However, micromolar concentrations of such compounds could be toxic due to their excessive accumulation in mitochondria. We studied possible pathophysiological effects of one such cation, i.e. dodecyltriphenylphosphonium (C(12)-TPP), in the yeast Saccharomyces cerevisiae. First, we found that C(12)-TPP induces high-amplitude mitochondrial swelling. The swelling can be prevented by addition of protonophorous uncoupler FCCP or antioxidant alpha-tocopherol, but not other tested antioxidants (N-acetylcysteine and Trolox). Second, FCCP prevents ROS-sensitive fluorescent dye (dichlorofluorescein diacetate) staining of yeast treated with C(12)-TPP. We also showed that all tested antioxidants partially restore the growth inhibited by C(12)-TPP. The latter points that ROS rather than the mitochondria swelling limit the growth rate.

Synthetic and natural polyanions induce cytochrome c release from mitochondria in vitro and in situ

A synthetic polyanion composed of styrene, maleic anhydride, and methacrylic acid (molar ratio 56:37:7) significantly inhibited the respiration of isolated rat liver mitochondria in a time-dependent fashion that correlated with 1) collapse of the mitochondrial membrane potential and 2) high amplitude mitochondrial swelling. The process is apparently Ca(2+) dependent. Since it is blocked by cyclosporin A, the process is ascribed to induction of the mitochondrial permeability transition. In mitoplasts, i.e., mitochondria lacking their outer membranes, the polyanion rapidly blocked respiration. After incubation of rat liver mitochondria with the polyanion, cytochrome c was released into the incubation medium. In solution, the polyanion modified by conjugation with fluorescein formed a complex with cytochrome c. Addition of the polyanion to cytochrome c-loaded phosphatidylcholine/cardiolipin liposomes induced the release of the protein from liposomal membrane evidently due to coordinated interplay of Coulomb and hydrophobic interactions of the polymer with cytochrome c. We conclude that binding of the polyanion to cytochrome c renders it inactive in the respiratory chain due to exclusion from its native binding sites. Apparently, the polyanion interacts with cytochrome c in mitochondria and releases it to the medium through breakage of the outer membrane as a result of severe swelling. Similar properties were demonstrated for the natural polyanion, tobacco mosaic virus RNA. An electron microscopy study confirmed that both polyanions caused mitochondrial swelling. Exposure of cerebellar astroglial cells in culture to the synthetic polyanion resulted in cell death, which was associated with nuclear fragmentation.

The role of mitochondrial palmitate/Ca2+-activated pore in palmitate-induced apoptosis

Evidence for the possible involvement of the mitochondrial cyclosporin A-insensitive palmitate/Ca(2+)-activated pore in the apoptotic process is presented. It has been established that the opening of palmitate/Ca(2+)-activated pore results in high-amplitude swelling of mitochondria and the release of apoptosis-induced factor from organelles. These processes are accompanied by a transitory small decrease of membrane potential, which recovers in 1 min. The possible role of palmitate/Ca(2+)-activated pore in the induction of palmitate-activated apoptosis is discussed.

Direct mitochondria permeabilization by a new anticancer agent erucylphosphohomocholine

Alkylphosphocholines are a new class of anticancer agents also representing a potential source for the treatment of tropical diseases caused by Leismania and Trypanosoma parasites. Most of the reported data suggest that mitochondria might be a main target, although direct mitochondrial effects of alkylphosphocholines have never been studied so far. In this work we show that erucylphosphohomocholine, a new antineoplastic alkylphosphocholine, permeabilized the inner membrane of isolated rat liver mitochondria, causing (i) a decrease of the inner membrane potential, (ii) an increase in state 4 respiration, (iii) a significant decrease in the rate of ATP synthesis, and (iv) low amplitude swelling of mitochondria at concentrations of 25–50 uM. In the presence of cyclosporin A, it strongly inhibited mitochondrial respiration. At 100 uM, erucylphosphohomocholine caused biphasic high amplitude swelling of mitochondria. On the other hand, oligomycin or cyclosporin A partially protected certain human cancer cell lines against apoptosis induced by this drug. Our findings demonstrate that direct mitochondrial alterations might be involved in anticancer and antiprotozoal activities of alkylphosphocholines. (Supported by DIME, National University of Colombia, Medellin Branch; by a grant from the Volkswagen‐Stiftung, Germany, VWZN2047; and Genzyme Pharmaceuticals (Liestal, Switzerland).

Identification and Characterization of Cannabinoids That Induce Cell Death through Mitochondrial Permeability Transition in Cannabis Leaf Cells

Cannabinoids are secondary metabolites stored in capitate-sessile glands on leaves of Cannabis sativa. We discovered that cell death is induced in the leaf tissues exposed to cannabinoid resin secreted from the glands, and identified cannabichromenic acid (CBCA) and Delta(1)-tetrahydrocannabinolic acid (THCA) as unique cell death mediators from the resin. These cannabinoids effectively induced cell death in the leaf cells or suspension-cultured cells of C. sativa, whereas pretreatment with the mitochondrial permeability transition (MPT) inhibitor cyclosporin A suppressed this cell death response. Examinations using isolated mitochondria demonstrated that CBCA and THCA mediate opening of MPT pores without requiring Ca(2+) and other cytosolic factors, resulting in high amplitude mitochondrial swelling, release of mitochondrial proteins (cytochrome c and nuclease), and irreversible loss of mitochondrial membrane potential. Therefore, CBCA and THCA are considered to cause serious damage to mitochondria through MPT. The mitochondrial damage was also confirmed by a marked decrease of ATP level in cannabinoid-treated suspension cells. These features are in good accord with those of necrotic cell death, whereas DNA degradation was also observed in cannabinoid-mediated cell death. However, the DNA degradation was catalyzed by nuclease(s) released from mitochondria during MPT, indicating that this reaction was not induced via a caspase-dependent apoptotic pathway. Furthermore, the inhibition of the DNA degradation only slightly blocked the cell death induced by cannabinoids. Based on these results, we conclude that CBCA and THCA have the ability to induce necrotic cell death via mitochondrial dysfunction in the leaf cells of C. sativa.

Synergistic effects of avicins and hydroperoxides on mitochondria: a possible mechanism for the selective killing of cancer cells

Avicins D and G are a class of plant saponins with pro-apoptotic activity in cancer cells, which is due in part to the direct perturbation of mitochondria (Haridas et al., PNAS USA, 2001). In this work, we studied the influence of avicins on the energy metabolism in normal rat liver mitochondria. Mitochondria, when treated with avicins, underwent a significant decrease in oxygen consumption in metabolic state 3 and in uncoupled state. The respiration was completely restored by addition of exogenous cytochrome c. Avicins also markedly increased the rotenone-insensitive oxidation of external NADH in the presence of exogenous cytochrome c, without induction of high amplitude swelling of mitochondria, thus confirming that the earlier effect of avicins is due to a permeabilization of the outer mitochondrial membrane to cytochrome c. On the other hand, avicins debilitate the enzymatic NAD(P)H-GSH-dependent antioxidant system of mitochondria due to a decrease of the inner membrane potential generation. As a result, avicins significantly increased a sensitivity of mitochondria to hydroperoxides, causing the acceleration of hydroperoxide-induced oxidation of endogenous NAD(P)H, the inner membrane potential drop and mitochondrial swelling. These data allow to explain relatively high selectivity of avicins in the killing of cancer cells, which have been shown to produce hydroperoxides with a very high rate.

Avicins, natural anticancer saponins, permeabilize mitochondrial membranes

Avicins are a class of natural saponins with selective pro-apoptotic activity in cancer cells. In this work, we studied the influence of avicins on metabolic state of rat liver mitochondria. Avicin-treated mitochondria underwent a significant decrease in oxygen consumption rate that was completely restored by addition of exogenous cytochrome c. On the other hand, avicins increased the rotenone-insensitive oxidation of external NADH in the presence of exogenous cytochrome c, long before high amplitude swelling of mitochondria was observed. The increase in external NADH oxidation was cyclosporin A-insensitive. Avicin G significantly accelerated hydroperoxide-induced oxidation of mitochondrial endogenous NAD(P)H, the drop of the inner membrane potential and the high amplitude swelling of mitochondria. The obtained data might explain selective induction of apoptosis in tumor cells by avicins. Based on other studies showing that tumor cells generate hydroperoxides with a very high rate, avicins could provide a new strategy of anticancer therapy by sensitizing cells with high levels of reactive oxygen species to apoptosis.

Structural determinants of fluorochemical-induced mitochondrial dysfunction.

Perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) are thought to induce peroxisome proliferation and interfere with mitochondrial metabolic pathways. Direct measurements revealed that PFOA and the unsubstituted sulfonamide of perfluorooctane (FOSA) uncouple mitochondrial respiration by increasing proton conductance. The purpose of this investigation was to characterize structural determinants responsible for the mitochondrial uncoupling effect of several structurally related fluorochemicals. Included in the study were PFOA, PFOS, FOSA, the N-acetate of FOSA (perfluorooctanesulfonamidoacetate, FOSAA), N-ethylperfluorooctanesulfonamide (N-EtFOSA), and the N-ethyl alcohol [2-(N-ethylperfluorooctanesulfonamido)ethyl alcohol, N-EtFOSE] and N-acetic acid (N-ethylperfluorooctanesulfonamidoacetate, N-EtFOSAA) of N-EtFOSA. Each test compound was dissolved in ethanol and added directly to an incubation medium containing substrate-energized rat liver mitochondria. Mitochondrial respiration and membrane potential were measured concurrently using an oxygen electrode and a TPP+ -selective electrode, respectively. All of the compounds tested, at sufficiently high concentrations, had the capacity to interfere with mitochondrial respiration, albeit via different mechanisms and with varying potencies. At sufficiently high concentrations, the free acids PFOA and PFOS caused a slight increase in the intrinsic proton leak of the mitochondrial inner membrane, which resembled a surfactant-like change in membrane fluidity. Similar effects were observed with the sulfonamide N-EtFOSE. Another fully substituted sulfonamide, N-EtFOSAA, at high concentrations caused inhibition of respiration, the release of cytochrome c, and high-amplitude swelling of mitochondria. The swelling was prevented by cyclosporin A or by EGTA, indicating that this compound induced the mitochondrial permeability transition. The unsubstituted and mono-substituted amides FOSA, N-EtFOSA, and FOSAA all exerted a strong uncoupling effect on mitochondria resembling that of protonophoric uncouplers. Among these compounds, FOSA was a very potent uncoupler of oxidative phosphorylation, with an IC50 of approximately 1 microM. These data suggest that the protonated nitrogen atom with a favorable pKa is essential for the uncoupling action of perfluorooctane sulfonamides in mitochondria, which may be critical to the mechanism by which these compounds interfere with mitochondrial metabolism to induce peroxisome proliferation in vivo.

Virus-induced permeability transition in mitochondria

Isolated rat liver mitochondria undergo permeability transition after supplementation with a suspension of tobacco mosaic virus. Four mitochondrial parameters proved the opening of the permeability transition pore in the inner mitochondrial membrane: increased oxygen consumption, collapse of the membrane potential, release of calcium ions from mitochondria, and high amplitude mitochondrial swelling. All virus-induced changes in mitochondria were prevented by cyclosporin A. These effects were not observed if the virus was treated with EGTA or disrupted by heating. Protein component of the virus particle in the form of 20S aggregate A-protein, or helical polymer, as well as supernatant of the heat-disrupted virus sample, had no effect on mitochondrial functioning. Electron microscopy revealed the direct interaction of the virus particles with isolated mitochondria. The possible role of the mitochondrial permeability transition pore in virus-induced apoptosis is discussed.

Ultrastructural changes of the vascular endothelium after intra-arterial administration of tumor necrosis factor-alpha (TNFalpha) in rat gliomas.

The ensuing ultrastructural changes in tumor vascular endothelial cells following intra-arterial administration of tumor necrosis factor-alpha (TNFalpha) were studied in an experimental rat glioma model. C6 glioma cells were implanted in Wistar rats and then after 14 days, 5 x 10(3) U of human natural-type TNFalpha (1.7 x 10(5) U/m2) was administered through the carotid artery. The animals were sacrificed at 3 or 24 h after TNFalpha treatment. A detailed examination with transmission electron microscope revealed swelling of the tumor vascular endothelial cell nuclei and mitochondria with matrix densities at 3 h. At 24 h, these cells demonstrated the presence of high amplitude mitochondrial swelling or the violent blebbing characteristic of damaged mitochondria; the cytoplasm was swollen enormously and there were dissolution of cytoplasmic organelles and rupture of the plasma membrane. The observed findings were typical of cell necrosis and confirms yet another mechanism by which TNFalpha exerts its anti-tumor effects, that is, necrotizing effects on tumor vascular endothelium. The information appears to be important in the context of clinical application of intra-arterial TNFalpha in the treatment of malignant gliomas.

Derivatives of 1,4-dihydropyridines as modulators of ascorbate-induced lipid peroxidation and high-amplitude swelling of mitochondria, caused by ascorbate, sodium linoleate and sodium pyrophosphate

A group of 26 2,6-dimethyl-3,5-disubstituted and 2,6-dimethyl-3,4, 5-trisubstituted-1,4-dihydropyridines (1,4-H(2)Py=1,4-DHPs) and five related pyridines were studied as inhibitors of rat liver mitochondrial swelling and O(2) uptake by ascorbic acid-dependent lipid peroxidation (LP) and as modulators of mitochondrial swelling induced by Na(+)-linoleate or Na(+)-pyrophosphate. 1,4-DHPs studied include 4-unsubstituted and 4-methyl- and 4-phenyl-substituted 3, 5-dialkoxycarbonylderivatives of 2,6-dimethyl-1,4-DHP with variations in alkoxy chain length and composition, 4-unsubstituted and 4-methyl-, 4-aryl- and 4-pyridyl-substituted 3, 5-dianilidocarbonylderivatives, and a structurally related group of 3,5-dipyridylamidocarbonylderivatives. Many 1,4-DHPs possess marked antioxidant (AO) and membrane stabilizing activity, expressed as the mitochondrial swelling (deltaA(520)/t) and/or O(2) uptake rate decrease (V(0)/V) as well as prolongation of the induction period (tau/tau(0)) of mitochondrial swelling and/or O(2) uptake at ascorbic acid-dependent LP of rat liver mitochondria. 4-Unsubstituted 3,5-dialkoxycarbonyl-2,6-dimethyl-1,4-DHPs, as well as 4-unsubstituted or those possessing lipophylic 4-aryl- groups 3, 5-diamido-2,6-dimethyl-1,4-DHPs, reveal marked AO and membrane stabilizing properties. Oxidized (heteroaromatized) derivatives have minimal activity. Perhaps 1,4-DHPs preferably act as antioxidants on stages of initiation and prolongation of LP chain reactions at low concentrations: IC(50) (when V(0)/V or tau/tau(0)=2) are 0.1 microM to 100 microM. At 100 microM 3,5-di-p-hydroxyphenoxycarbonyl- and 3, 5-di-p-tolyloxycarbonyl-2,6-dimethyl-1,4-DHPs, as well as 3, 5-diethoxycarbonyl-2,6-dimethylpyridine (oxidized form of Hantzsch ester) and 3,5-diamyloxycarbonyl-2,6-dimethylpyridine, alter the mitochondrial swelling rate in the presence of natural protonophore Na(+)-linoleate (0.063 mM and 0.125 mM). 3,5-Di-n-butyloxycarbonyl-2, 6-dimethyl-1,4-DHP at 100 microM completely stops mitochondrial swelling in the presence of 0.8 mM Na(+)-pyrophosphate. In the presence of many of the 1,4-DHPs, the lipid peroxidation process was inhibited. However, the swelling process could be prolonged, promoted, accelerated or inhibited-depending on 1,4-DHPs structure, concentration, the type of initiators of the swelling process and the medium composition.

Maternal antioxidant treatments prevent diabetes-induced alterations of mitochondrial morphology in rat embryos.

Previous studies have suggested that production of reactive oxygen species by embryonic mitochondria may have a role in the induction of both high-amplitude mitochondrial swelling and embryonic dysmorphogenesis in diabetic pregnancy. The present study analyzed the relationships between a putative metabolite-induced production of free oxygen radicals, mitochondrial lipid peroxidation, and high-amplitude mitochondrial swelling in embryos during organogenesis. For studies in vitro, day 9 embryos of normal rats were cultured for 48 h with a high concentration of glucose in the absence or presence of alpha-cyano-4-hydroxycinnamic acid (CHC), a mitochondrial pyruvate transport inhibitor. The morphology of mitochondria in the neuroepithelium of the embryos was studied with the aid of transmission electron microscopy. For studies in vivo, normal and diabetic pregnant rats were fed a diet supplemented with the antioxidants alpha-tocopherol (vitamin E) or 2,6-di-tert-butyl-4-methylphenol (BHT), and the ultrastructure of mitochondria in the embryonic neuroepithelium and in the visceral yolk sac was investigated on gestational day 11. Exposure to a high concentration of glucose in vitro or to maternal diabetes in vivo induced high-amplitude swelling of mitochondria in the neuroepithelium of the embryos. The swelling of mitochondria was prevented by addition of CHC to the culture media or by maternal ingestion of antioxidant-supplemented food. In diabetic pregnancy, embryonic mitochondria during organogenesis produce free oxygen radicals that cause mitochondrial lipid peroxidation and swelling and furthermore embryonic dysmorphogenesis. Dietary supplementation with antioxidants to the mother may prevent embryonic malformations in diabetic pregnancy by inhibition of mitochondrial dysfunction.

Altered mitochondrial morphology of rat embryos in diabetic pregnancy

Previous studies in vivo and in vitro have suggested that the oxidative metabolism of the embryo may have a role in the teratogenicity of diabetic pregnancy. In particular, the production of reactive oxygen species by the embryonic mitochondria has been implicated in the teratological process. The induction of congenital malformations by the diabetic milieu occurs during the early embryonic development. The present study aimed to estimate the role of the embryonic mitochondria in the teratological process of diabetic pregnancy by studying mitochondrial morphology in the embryos exposed to a diabetic environment in vivo or in vitro during early organogenesis and late fetal development. For studies in vivo embryos of control or streptozotocin-diabetic rats were taken at gestational days 9-11 and subjected to light and electron microscopical analysis. The brain, heart, and liver of day-15 fetuses were also observed. For studies in vitro day-9 embryos of normal rats were cultured in a whole-embryo culture system for 48 hours. The culture media were supplied with high concentration of diabetes-related substrates and metabolites, and their effect on structure of embryonic neuroepithelial cells determined. The light microscopical observations demonstrated numerous cytoplasmic vacuoles in the ectoderm of day-9 embryos and in the neuroepithelium and blood cells of day-10 and day-11 embryos of diabetic rats. Ultrastructurally, these vacuoles were found to be mitochondria undergoing large-amplitude swelling with loss of matrix density and disturbed cristae. In contrast, no mitochondrial differences were found in the brain, heart, and liver, when day-15 fetuses from normal and diabetic rats were compared. Ultrastructural analysis of day-9 embryos cultured for 48 hours in the presence of high concentrations of D-glucose, pyruvate, beta-hydroxybutyrate, and alpha-ketoisocaproate also showed high-amplitude mitochondrial swelling in the neuroepithelium. The mitochondrial swelling was, however, not found in embryos cultured in a high concentration of L-glucose, excluding simple osmotic effects of the diabetes-related substrates and metabolites. The mitochondrial morphological changes appeared in embryos subjected to a diabetic environment during a time period when the congenital malformations in diabetic pregnancy are induced. The results support the notion that embryonic mitochondria are involved in the teratological process of diabetic pregnancy.