Isolated Rat Liver Research Articles

Anthocyanin-Rich Extract of Red Cabbage Attenuates Advanced Alcohol Hepatotoxicity in Rats in Association with Mitochondrial Activity Modulation

Abstract Aim The liver is the main target for alcohol-induced injury. The aims of this work were to carry out further research into the mechanisms of liver damage induced by long-term administration of high-dose ethanol to rats and to evaluate the hepatoprotective potential of red cabbage (Brassica oleracea var. capitata f. rubra) anthocyanins (RCE). Material/Methods Male albino Wistar rats were divided into four groups. Group 1 was the control. Groups 2 through 4 received ethanol (4 g/kg body weight, 8 weeks). Group 3 received 11 mg RCE/kg and Group 4 received 22 mg RCE/kg. Dry lyophilised RCE was prepared from fresh red cabbage and analysed. We then evaluated the liver histology, mitochondrial respiration, and biochemical and immunological parameters in these groups of rats. Results The rat intoxication caused steatohepatitis characterised by macro- and microvesicular steatosis, ballooning and fatty liver dystrophies, lymphocytic infiltration, neutral lipid accumulation, and elevations of the serum activities of the liver injury marker enzymes. The treatment of alcohol-administered rats with RCE (mainly, cyanidin-3-diglucoside-5-glucoside, cyanidin-3-coumaroylrutinoside-5-hexoside, cyanidin-3-feruloylrutinoside-5-hexoside, delphinidin-3-feruloylrutinoside-hexoside) dose-dependently alleviated these pathological changes: The sizes of hepatocyte lipid vacuoles and the inflammatory signs were decreased, and the levels of the rat serum biochemical markers of liver injury, proinflammatory cytokines TNFα and IL-6, and the adipokine leptin (and not TGFβ) were significantly reversed. The RCE administration during intoxication completely recovered the changed liver mitochondria respiration rates and the ADP/O coefficient, as well as the phagocytic index and neutrophil metabolic activity in the blood. In experiments in vitro, RCE (13.6 μg/ml) modulated the respiratory parameters of isolated rat liver mitochondria, dissipated mitochondrial membrane potential, and inhibited the Ca2+-induced mitochondrial permeability transition. Conclusion The red cabbage anthocyanins could be useful for treatment of alcoholic liver injury due to their Ca2+-ionophoric/protonophoric activity, influence on Ca2+ homeostasis, and improving mitochondrial functions and inflammatory status.

Complexations of β-cyclodextrins with naringenin, naringin and catechin: thermodynamic parameters and regulation of mitochondrial functions in vitro

ABSTRACT Flavonoids are exogenous phytochemicals with high antioxidative potential. The present work provides the mechanism of the complexations of low water-soluble flavanone naringenin, its glycosylated form naringin, and water-soluble flavan-3-ol catechin with β-cyclodextrin (βCD) and hydroxypropyl-β-cyclodextrin (HPβCD). The flavonoids studied showed high affinity in the interactions with HPβCD (the association constants K and the stoichiometry N were equal to 8500 ± 950 М−1 and 0.85 ± 0.05, 9400 ± 1200 М−1 and 1.7 ± 0.10, 7550 ± 600 M−1 and 1.25 ± 0.05 in the case of naringenin, naringin and catechin, respectively). The formations of the complexes with β-cyclodextrins markedly increased the water solubility of naringenin and naringin. The flavonoid–HPβСD complexations were exothermic (ΔH ˂ 0) and spontaneous (ΔG ˂ 0). The naringenin and naringin complexations with HPβCD were entropy-driven, and the catechin complexation was enthalpy-driven. Flavonoid molecules dispersed in the HPβCD structure during complexations. The flavonoid–HPβCD associations demonstrated enthalpy–entropy compensation. Naringenin (5–50 μM) and, to a lesser extent, catechin inhibited the respiration of isolated rat liver mitochondria. Naringenin, naringin and catechin (5–25 μM) increased the rate of Ca2+-induced mitochondrial permeability transition. The complexations with HPβCD did not significantly influence the effects of the flavonoids on respiration and enhanced the effects on mitochondrial permeability transition.

Alkyl esters of 7-hydroxycoumarin-3-carboxylic acid as potent tissue-specific uncouplers of oxidative phosphorylation: Involvement of ATP/ADP translocase in mitochondrial uncoupling

An impressive body of evidence has been accumulated now on sound beneficial effects of mitochondrial uncouplers in struggling with the most dangerous pathologies such as cancer, infective diseases, neurodegeneration and obesity. To increase their efficacy while gaining further insight in the mechanism of the uncoupling action has been remaining a challenge. Encouraged by our previous promising results on lipophilic derivatives of 7-hydroxycoumarin-4-acetic acid (UB-4 esters), here, we use a 7-hydroxycoumarin-3-carboxylic acid scaffold to synthesize a new series of 7-hydroxycoumarin (umbelliferone, UB)-derived uncouplers of oxidative phosphorylation – alkyl esters of umbelliferone-3-carboxylic acid (UB-3 esters) with varying carbon chain length. Compared to the UB-4 derivatives, UB-3 esters proved to be stronger uncouplers: the most effective of them caused a pronounced increase in the respiration rate of isolated rat heart mitochondria (RHM) at submicromolar concentrations. Both of these series of UB derivatives exhibited a striking difference between their uncoupling patterns in mitochondria isolated from liver and heart or kidney, namely: a pronounced but transient decrease in membrane potential, followed by its recovery, was observed after the addition of these compounds to isolated rat liver mitochondria (RLM), while the depolarization of RHM and rat kidney mitochondria (RKM) was rather stable under the same conditions. Interestingly, partial reversal of this depolarization in RHM and RKM was caused by carboxyatractyloside, an inhibitor of ATP/ADP translocase, thereby pointing to the involvement of this mitochondrial membrane protein in the uncoupling activity of both UB-3 and UB-4 esters. The fast membrane potential recovery in RLM uncoupled by the addition of the UB esters was apparently associated with hydrolysis of these compounds, catalyzed by mitochondrial aldehyde dehydrogenase (ALDH2), being in high abundance in liver compared to other tissues. Protonophoric properties of the UB derivatives in isolated mitochondria were confirmed by measurements of RHM swelling in the presence of potassium acetate. In model bilayer lipid membranes (liposomes), proton-carrying activity of UB-3 esters was demonstrated by measuring fluorescence response of the pH-dependent dye pyranine. Electrophysiological experiments on identified neurons from Lymnaea stagnalis demonstrated low neurotoxicity of UB-3 esters. Resazurin-based cell viability assay showed low toxicity of UB-3 esters to HEK293 cells and primary human fibroblasts. Thus, the present results enable us to consider UB-3 esters as effective tissue-specific protonophoric mitochondrial uncouplers.

A pan-tumor-siRNA aptamer chimera to block nonsense-mediated mRNA decay inflames and suppresses tumor progression

Immune-checkpoint blockade (ICB) therapy has changed the clinical outcome of many types of aggressive tumors, but there still remain many cancer patients that do not respond to these treatments. There is an unmet need to develop a feasible clinical therapeutic platform to increase the rate of response to ICB. Here we use a previously described clinically tested aptamer (AS1411) conjugated with SMG1 RNAi (AS1411-SMG1 aptamer-linked siRNA chimeras [AsiCs]) to inhibit the nonsense-mediated RNA decay pathway inducing tumor inflammation and improving response to ICB. The aptamer AS1411 shows binding to numerous mouse and human tumor cell lines tested. AS1411 induces tumor cytotoxicity in long incubation times, which allows for the use of the aptamer as a carrier to target the RNAi inhibition to the tumor. The AS1411-SMG1 AsiCs induce a strong antitumor response in local and systemic treatment in different types of tumors. Finally, AS1411-SMG1 AsiCs are well tolerated with no detected side effects.

Inhibition of respiratory complex I by 6-ketocholestanol: Relevance to recoupling action in mitochondria

6-Ketocholestanol (kCh) is known as a mitochondrial recoupler, i.e. it abolishes uncoupling of mitochondria by such potent agents as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 3,5-di(tert-butyl)-4-hydroxybenzylidenemalononitril (SF6847) [Starkov et al., 1997]. Here, we report data on the kCh-induced inhibition of both NADH-oxidase and NADH-ubiquinone oxidoreductase activities of the respiratory complex I in bovine heart submitochondrial particles (SMP). Based on the absence of such inhibition with hexaammineruthenium (III) (HAR) as the complex I electron acceptor, the kCh effect could be associated with the ubiquinone-binding centre of this respiratory enzyme. In isolated rat liver mitochondria (RLM), kCh inhibited oxygen consumption with the glutamate/malate, substrates of NAD-linked dehydrogenases, while no inhibition of RLM respiration was observed with succinate, in agreement with the absence of the kCh effect on the succinate oxidase activity in SMP. Three kCh analogs (cholesterol, 6α-hydroxycholesterol, and 5α,6α-epoxycholesterol) exhibited no effect on the NADH oxidase activities in both SMP and RLM. Importantly, the kCh analogs were ineffective in the recoupling of RLM treated with CCCP or SF6847. Therefore, interaction of kCh with the complex I may be involved in the kCh-mediated mitochondrial recoupling.

Complexation potentiated promising anti‐diabetic and anti‐oxidative synergism between ZN(ii) and ferulic acid: A multimode study

This study was done to investigate the anti-diabetic and anti-oxidative synergism between zinc(II) and ferulic acid through complexation. Zinc sulphate was complexed with ferulic acid in a 1:2 molar ratio. The complex was characterized using Fourier-transform infrared spectroscopy, proton NMR and high-resolution mass spectroscopy techniques and evaluated for cellular toxicity. In silico, in vitro, cell-based and tissue experimental models were used to test the anti-diabetic and anti-oxidant activities of the complex relative to its precursors. A zinc(II)-biferulate.2H2 O complex was formed. The in vitro radical scavenging, anti-lipid peroxidative and α-glucosidase and α-amylase inhibitory activity of the complex was 1.7-2.1 folds more potent than ferulic acid. Zn(II) complexation increased the anti-glycation activity of ferulic acid by 1.5 folds. The complex suppressed lipid peroxidation (IC50 =48.6 and 331 μM) and GHS depletion (IC50 =33.9 and 33.5μM) in both Chang liver cells and isolated rat liver tissue. Its activity was 2.3-3.3 folds more potent than ferulic acid and statistically comparable to ascorbic acid. Zn(II) complexation afforded ferulic acid improved glucose uptake activity in L-6 myotube (EC50 =11.7 vs. 45.7μM) and isolated rat muscle tissue (EC50 =501 and 1510 μM). Complexation increased muscle tissue zinc(II) uptake and hexokinase activity. Docking scores of the complex (-7.24 to -8.25 kcal/mol) and ferulic acid (-5.75 to 6.43 kcal/mol) suggest the complex had stronger interaction with protein targets related to diabetes, which may be attributed to the 2 ferulic acid moieties and Zn(II) in the complex. Moreover, muscle tissue showed increased phospho-Akt/pan-Akt ratio upon treatment with complex. The complex was not hepatotoxic and myotoxic at in vitro cellular level. Zn(II) complexation may be promising therapeutic approach for improving the glycaemic control and anti-oxidative potential of natural phenolic acids.

Mitotherapy in doxorubicin induced cardiotoxicity: A promising strategy to reduce the complications of treatment

AimsDoxorubicin is a potent and broad-spectrum antineoplastic medication prescribed for both solid and hematological malignancies. Despite its value, the clinical use of doxorubicin is limited due to cardio-oncologic complication and cardiotoxic adverse effect. Among the mechanisms proposed for its toxicity, mitochondrial dysfunction has gained more attention. Therefore, if damaged mitochondria are replaced by normal efficient mitochondria, cardiac toxicity is expected to be reduced or improved. In this way, we have studied the efficiency of transplantation of freshly isolated rat liver mitochondria in neonatal rat cardiomyocytes that have been damaged by doxorubicin. Materials and methodsFor this purpose, isolated mitochondria were characterized using mitochondrial complex II, membrane potential and swelling evaluations, and also fluorescence and electron microscopy. Afterward, the effect of mitotherapy on the damaged cardiomyocytes was investigated by using annexin V/PI staining, MTT, ROS, MMP, lipid peroxidation, GSH and ATP evaluations. Key findings and significanceTransplanted mitochondria could remarkably enter the neonatal rat cardiomyocytes. Addition of mitochondria to the damaged cardiomyocytes, significantly increased cell viability by reducing the level of reactive oxygen species and lipid peroxidation, increasing of ∆Ψ, ATP and GSH contents and decreasing of apoptotic and necrotic cell death. Our results showed that mitotherapy has a significant restorative effect on cardiotoxicity induced by doxorubicin, which promises a better future to reduce the complications of cancer treatment.

Subnormothermic isolated organ perfusion with Nicorandil increased cold ischemic tolerance of liver in experimental model.

The cold ischemia -reperfusion injury may lead to microcirculatory disturbances, hepatocellular swelling, inflammation, and organ dysfunction. Nicorandil is an anti-ischemic, ATP-sensitive potassium (KATP) channel opener drug and has proved its effectiveness against hepatic Ischemia/Reperfusion (I/R) injury. This study aimed to investigate the effect of Nicorandil on mitochondrial apoptosis, oxidative stress, inflammation, histopathological changes, and cold ischemic tolerance of the liver in an ex vivo experimental isolated-organ-perfusion model. We used an ex vivo isolated rat liver perfusion system for this study. The grafts were retrieved from male Wistar rats (n = 5 in each), preserved in cold storage (CS) for 2 or 4 hours (group 1, 2), or perfused for 2 or 4 hours (group 3, 4) immediately after removal with Krebs Henseleit Buffer (KHB) solution or Nicorandil containing KHB solution under subnormothermic (22-25°C) conditions (group 5, 6). After 15 minutes incubation at room temperature, the livers were reperfused with acellular, oxygenated solution under normothermic condition for 60 minutes. In the Nicorandil perfused groups, significantly decreased liver enzymes, GLDH, TNF-alpha, and IL-1ß were measured from the perfusate. Antioxidant enzymactivity was higher in the perfused groups. Histopathological examination showed ameliorated tissue deterioration, preserved parenchymal structure, decreased apoptosis, and increased Bcl-2 activity in the Nicorandil perfused groups. Perfusion with Nicorandil containing KHB solution may increase cold ischemic tolerance of the liver via mitochondrial protection which can be a potential therapeutic target to improve graft survival during transplantation.

Synthesis, in silico prediction of sites of metabolism and in-vitro hepatotoxicity evaluationofnew seriesN’-substituted 3-(1,3,7-trimethyl-xanthin-8-ylthio)propanehydrazides

New series of 3-(1,3,7-trimethyl-xanthin-8-ylthio)propanehydrazide derivatives were designed and synthesized. The targed compounds were obtained in yields of 54 to 100% and their structures were elucidated by FTIR,1H NMR,13C NMR, MS and microanalyses. The tested compounds were subjected to in silico prediction of sites of metabolism (SOMs). The predictions show thatthe main metabolic changes will be primarily related to oxidation of the sulfur atom in the side chain, carried out under the action of CYP2C19, as well as O-demethylation of compounds containing methoxy groups.The N-demethylation of the xanthine fragment was determined to be regulated by CYP1A2, CYP2C9, CYP2D6 and CYP3A4. Theperformed in vitro studies confirmed for two of the tested compounds to be low hepatotoxic, due to the presented prooxidant effects at subcellular level (isolated rat liver microsomes). These results highlight these molecules as promising hydrazide-hydrazone structures for the design of compounds with low hepatotoxicity.

The rapid transformation of triclosan in the liver reduces its effectiveness as inhibitor of hepatic energy metabolism

Triclosan (5-chloro-2'-[2,4-dichlorophenoxi]-phenol) is a polychlorinated biphenolic antimicrobial, utilized as antiseptic and preservative in hygiene products and medical equipment. Triclosan causes mitochondrial dysfunction (uncoupling, inhibition of electron flow), as demonstrated in isolated rat liver mitochondria. These actions in the mitochondria could compromise energy-dependent metabolic fluxes in the liver. For this reason, the present work aimed at investigating how these effects on isolated mitochondria translate to the whole and intact hepatocyte. For accomplishing this, the isolated perfused rat liver was utilized, a system that preserves both microcirculation and the cell-to-cell interactions. In addition, the single-pass triclosan hepatic transformation was also evaluated by HPLC as well as the direct action of triclosan on gluconeogenic enzymes. The results revealed that triclosan decreased anabolic processes (e.g., gluconeogenesis) and increased catabolic processes (e.g., glycolysis, ammonia output) in the liver, generally with a complex pattern of concentration dependences. Unlike the effects on isolated mitochondria, which occur in the micromolar range, the effects on intact liver required the 10−5 to 10−4 M range. The most probable cause for this behavior is the very high single-pass transformation of triclosan, which was superior to 95% at the portal concentration of 100 μM. The concentration gradient along the sinusoidal bed is, thus, very pronounced and the response of the liver reflects mainly that of the periportal cells. The high rates of hepatic biotransformation may be a probable explanation for the low acute toxicity of triclosan upon oral ingestion.

Alkyl esters of umbelliferone-4-acetic acid as protonophores in bilayer lipid membranes and ALDH2-dependent soft uncouplers in rat liver mitochondria

A great variety of coumarin-related compounds, both natural and synthetic, being often brightly fluorescent, have shown themselves beneficial in medicine for both therapeutic and imaging purposes. Here, in search for effective uncouplers of oxidative phosphorylation, we synthesized a series of 7-hydroxycoumarin (umbelliferone, UB) derivatives combining rather high membrane affinity with the presence of a hydroxyl group deprotonable at physiological pH - alkyl esters of umbelliferone-4-acetic acid (UB-4 esters) differing in alkyl chain length. Addition of UB-4 esters to isolated rat liver mitochondria (RLM) resulted in their rapid depolarization, unexpectedly followed by membrane potential recovery on a minute time scale. According to TLC and HPLC data, incubation of RLM with UB-4 esters caused their hydrolysis, which led to disappearance of the uncoupling activity (recoupling). Both mitochondrial recoupling and hydrolysis of UB-4 esters were suppressed by inhibitors of mitochondrial aldehyde dehydrogenase (ALDH2), disulfiram and daidzin, thus pointing to the involvement of this enzyme in the recoupling of RLM incubated with UB-4 esters. The protonophoric mechanism of mitochondrial uncoupling by UB-4 esters was proved in experiments with artificial bilayer lipid membranes (BLM): these compounds induced proton-selective electrical current across planar BLM and caused dissipation of pH gradient on liposomes. UB-4 esters showed antibacterial activity against Bacillus subtilis, Staphylococcus aureus and Mycobacterium smegmatis.

Assessment of the effects of Annona muricata leaf aqueous extract in vitro

Background: The extracts of Annona muricata, also known as graviola, form part of traditional medicine.
 Objectives: Verification of their effects and elucidation of their mechanisms is beneficial in terms of the utility of these extracts in treatment or prevention.
 Methods: We monitored the effectiveness of an extract of dried graviola leaves available for direct consumption on cancer cell lines and isolated rat liver mitochondria.
 Results: The Jurkat cell line exhibited the highest, sensitivity, with the metabolic activity of cells reaching less than 10% at the highest tested concentration. The effect was dose-dependent. The viability of Jurkat cells was affected after more than the 72 hours. At the same time, there was also a reduction of mitochondrial membrane potential. At 24 to 48 hours incubation, no increase was found in the activity of caspase-3 but increased levels were found for the non-phosphorylated form of Bcl-2. Higher concentrations of graviola affected the mitochondrial redox state by dramatically reducing the levels of reduced glutathione. This was achieved through reduction in the activity of glutathione-S-transferase.
 Conclusion: Our findings indicate that the studied preparation of graviola displayed cytotoxic effects on some cells, depending on the dose and length of action.

In vitro toxic interaction of arsenic and hyperglycemia in mitochondria: an important implication of increased vulnerability in pre-diabetics.

Environmental pollutants and lifestyle both contribute to the rapidly increasing prevalence of type 2 diabetes mellitus (T2DM) worldwide. Evidence suggests that exposure to environmental contaminants such as arsenic is associated with impaired glucose metabolism and insulin signaling. In the present study, isolated rat liver mitochondria (1mg/ml) were co-exposed to low concentration of arsenic trioxide (ATO) ( IC25 = 40µM) and hyperglycemic condition (20, 40, 80, 160mM glucose or 20, 40, 80, 160mM pyruvate (PYR)). Mitochondrial dehydrogenase activity (complex II), glutathione content (GSH), reactive oxygen species (ROS), lipid peroxidation, mitochondrial membrane potential (ΔΨ), and mitochondrial swelling were then evaluated in the presence of ATO 40µM and PYR 40mM. Unexpectedly, glucose alone (20, 40, 80, 160mM) had no toxic effect on mitochondria, even at very high concentrations and even when combined with ATO. Interestingly, PYR at low concentrations (≤ 10mM) has a protective effect on mitochondria, but at higher concentrations (≥ 40mM) with ATO, it decreased the complex II activity and increased mitochondrial ROS production, lipid peroxidation, GSH depletion, mitochondrial membrane damage, and swelling (p < 0.05). In conclusion, PYR but not glucose increased ATO mitochondrial toxicity even at low concentrations. These results suggest that pre-diabetics with non-clinical hyperglycemia, who are inevitably exposed to low concentrations of arsenic through food and water, may develop mitochondrial dysfunction that accelerates their progression to diabetes over time.

5-Aminolevulinate improves metabolic recovery and cell survival of the liver following cold preservation.

Rationale: Hibernating thirteen-lined ground squirrels (GS; Ictidomys tridecemlineatus) are naturally adapted to prolonged periods of ultraprofound hypothermia (body temperature < 5 ºC) during torpor, and drastic oscillations of body temperature and ischemia/reperfusion-like stress during their short euthermic interbout arousals. Thus, their superior adaptability may hold tremendous promise for the advancement of donor organ cold preservation and subsequent organ transplantation. However, bridging hibernation research and translational medicine has been impeded by a dearth of in vitro research tools, till the recent establishment of the GS induced pluripotent stem cells (iPSCs). In this study, we reported the generation of functional hepatocyte-like cells (HLCs) from GS iPSCs. As temperature and oxygen supply affect cellular metabolism, we hypothesized that the GS HLCs can metabolically counter drastic temperature and oxygen supply changes. Differentially regulated metabolites can be evaluated and included into the preservation solution to mitigate temperature and ischemia/reperfusion-associated damage to donor livers.Methods: A protocol has been developed to produce GS iPSCs-derived HLCs. Comparative metabolomic analysis on GS HLCs and human donor liver samples revealed changes in metabolites caused by cold storage and rewarming. Human embryonic stem cell (ESC)-derived HLCs and ex vivo cold preservation and reperfusion of isolated rat livers were used to assess candidate metabolites that may have protective effects against preservation-related injuries.Results: GS iPSCs were efficiently differentiated into expandable, cryopreservation-compatible and functional HLCs. Metabolomic analysis unveiled distinct changes of mitochondrial metabolites between GS and human cells following cold storage and rewarming. GS and human HLC-based experiments indicated that the metabolism of 5-aminolevulinate (5-ALA) is key to restricting free radical production during rewarming. Survival of human HLCs was significantly increased following cold exposure and rewarming, as supplemented 5-ALA enhanced Complex III activity and improved mitochondrial respiration. Further, 5-ALA mitigated damage in rat livers following 48-h cold preservation and ex vivo reperfusion. Metabolomic and transcriptomic analyses revealed that supplemented 5-ALA promoted both anabolic and catabolic activities while alleviating cell death, inflammation, hypoxia and other stress responses in isolated perfused rat livers.Conclusion: In the liver, rewarming from ultraprofound hypothermia imposes complex metabolic challenges and stresses on the mitochondria. Metabolites such as 5-ALA can help alleviate mitochondrial stress. Supplementing 5-ALA to the liver preservation solution can substantially improve the functional recovery of rat livers following prolonged cold preservation, rewarming and reperfusion.

Structural and functional changes in rat liver mitochondria under calcium ion loading in the absence and presence of flavonoids

The aim of the present work was to elucidate the mechanisms of calcium ion-induced impairments of the ultrastructure and functional activity of isolated rat liver mitochondria in the absence and presence of a number of flavonoids in vitro. In the presence of exogenous Ca²⁺ (20-60 μM), mitochondrial heterogeneity in size and electron density markedly increased: most organelles demonstrated a swollen electron-light matrix, bigger size, elongated cristae and a reduced their number, a damaged native structure of the inner membrane up to its detachment, and some mitochondria showed a more electron-dense matrix (condensed mitochondria). The calcium-induced opening of the mitochondrial permeability transition pores (MPTP) resulted in the ultrastructural disturbances and in the effective inhibition of the respiratory activity of rat liver mitochondria. The flavonoids (10-25 μM) naringenin and catechin, dose-dependently inhibited the respiratory activity of mitochondria and stimulated the MPTP opening in the presence of Ca²⁺ ions. Since Ruthenium red, an inhibitor of the mitochondrial Ca²⁺ uniporter, effectively prevented Ca²⁺-induced MPTP opening both in the absence and presence of flavonoids, we hypothesized that the effect of flavonoids on the MPTP opening could be mediated by stimulation of the Ca²⁺ uniporter.

Age-Dependent Changes in the Function of Mitochondrial Membrane Permeability Transition Pore in Rat Liver Mitochondria

Mitochondria play an important role in the cell aging process. Changes in calcium homeostasis and/or increased reactive oxygen species (ROS) production lead to the opening of mitochondrial permeability transition pore (MPTP), depolarization of the inner mitochondrial membrane, and decrease of ATP production. Our work aimed to monitor age-related changes in the Ca2+ ion effect on MPTP and the ability of isolated rat liver mitochondria to accumulate calcium. The mitochondrial calcium retention capacity (CRC) was found to be significantly affected by the age of rats. Measurement of CRC values of the rat liver mitochondria showed two periods when 3 to17-week old rats were tested. 3-week and 17-week old rats showed lower CRC values than 7-week old animals. Similar changes were observed while testing calcium-induced swelling of rat liver mitochondria. These findings indicate that the mitochondrial energy production system is more resistant to calcium-induced MPTP opening accompanied by the damaging effect of ROS in adult rats than in young and aged animals.

Comprehensive understanding of multiple actions of anticancer drug tamoxifen in isolated mitochondria

Tamoxifen has been widely used in the treatment of estrogen receptor (ER)-positive breast cancer, whereas it also exhibits ER-independent anticancer effects in various cancer cell types. As one of the convincing mechanisms underlying the ER-independent effects, induction of apoptosis through mitochondrial dysfunction has been advocated. However, the mechanism of action of tamoxifen even at the isolated mitochondrial level is not fully understood and remains controversial. Here, we attempted to comprehensively understand tamoxifen's multiple actions in isolated rat liver mitochondria through not only revisiting the actions hitherto reported but also conducting originally designed experiments. Using submitochondrial particles, we found that tamoxifen has potential as an inhibitor of both respiratory complex I and ATP synthase. However, these inhibitory effects were not elicited in intact mitochondria, likely because penetration of tamoxifen across the inner mitochondrial membrane is highly restricted owing to its localized positive charge (-N+H(CH3)2). This restricted penetration may also explain why tamoxifen is unable to function as a protonophore-type uncoupler in mitochondria. Moreover, tamoxifen suppressed opening of the mitochondrial permeability transition pore induced by Ca2+ overload through enhancing phosphate uptake into the matrix. The photoaffinity labeling experiments using a photolabile tamoxifen derivative (pTAM1) indicated that pTAM1 specifically binds to voltage-dependent anion channels (VDACs) 1 and 3, which regulate transport of various substances into mitochondria. The binding of tamoxifen to VDAC1 and/or VDAC3 could be responsible for the enhancement of phosphate uptake. Taking all the results together, we consider the principal impairment of mitochondrial functions caused by tamoxifen.

New Properties and Mitochondrial Targets of Polyphenol Agrimoniin as a Natural Anticancer and Preventive Agent.

Agrimoniin is a polyphenol from the group of tannins with antioxidant and anticancer activities. It is assumed that the anticancer action of agrimoniin is associated with the activation of mitochondria-dependent apoptosis, but its mitochondrial targets have not been estimated. We examined the direct influence of agrimoniin on different mitochondrial functions, including the induction of the mitochondrial permeability transition pore (MPTP) as the primary mechanism of mitochondria-dependent apoptosis. Agrimoniin was isolated from Agrimonia pilosa Ledeb by multistep purification. The content of agrimoniin in the resulting substance reached 80%, as determined by NMR spectroscopy. The cytotoxic effect of purified agrimoniin was confirmed on the cultures of K562 and HeLa cancer cells by the MTT assay. When tested on isolated rat liver mitochondria, agrimoniin at a low concentration (10 µM) induced the low-amplitude swelling, which was inhibited by the MPTP inhibitors ADP and cyclosporine A, activated the opening of MPTP by calcium ions and stimulated the respiration supported by succinate oxidation. Also, agrimoniin reduced the electron acceptor DCPIP in a concentration-dependent manner and chelated iron ions. Owing to all these properties, agrimoniin can stimulate apoptosis or activate mitochondrial functions, which can be helpful in the prevention and elimination of stagnant pathological states.

Rotating Magnetic Fields Inhibit Mitochondrial Respiration, Promote Oxidative Stress and Produce Loss of Mitochondrial Integrity in Cancer Cells.

Electromagnetic fields (EMF) raise intracellular levels of reactive oxygen species (ROS) that can be toxic to cancer cells. Because weak magnetic fields influence spin state pairing in redox-active radical electron pairs, we hypothesize that they disrupt electron flow in the mitochondrial electron transport chain (ETC). We tested this hypothesis by studying the effects of oscillating magnetic fields (sOMF) produced by a new noninvasive device involving permanent magnets spinning with specific frequency and timing patterns. We studied the effects of sOMF on ETC by measuring the consumption of oxygen (O2) by isolated rat liver mitochondria, normal human astrocytes, and several patient derived brain tumor cells, and O2 generation/consumption by plant cells with an O2 electrode. We also investigated glucose metabolism in tumor cells using 1H and 13C nuclear magnetic resonance and assessed mitochondrial alterations leading to cell death by using fluorescence microscopy with MitoTracker™ and a fluorescent probe for Caspase 3 activation. We show that sOMF of appropriate field strength, frequency, and on/off profiles completely arrest electron transport in isolated, respiring, rat liver mitochondria and patient derived glioblastoma (GBM), meningioma and diffuse intrinsic pontine glioma (DIPG) cells and can induce loss of mitochondrial integrity. These changes correlate with a decrease in mitochondrial carbon flux in cancer cells and with cancer cell death even in the non-dividing phase of the cell cycle. Our findings suggest that rotating magnetic fields could be therapeutically efficacious in brain cancers such as GBM and DIPG through selective disruption of the electron flow in immobile ETC complexes.

Evaluation of mitochondrial dysfunction due to oxidative stress in therapeutic, toxic and lethal concentrations of tramadol.

Tramadol (TR) is a centrally acting analgesic drug that is used to relieve pain. The therapeutic (0.1-0.8mg/l), toxic (1-2mg/l) and lethal (>2mg/l) ranges were reported for TR. The present study was designed to evaluate which doses of TR can induce liver mitochondrial toxicity. Mitochondria were isolated from the five rats' liver and were incubated with therapeutic to lethal concentrations (1.7-600μM) of TR. Biomarkers of oxidative stress including: reactive oxygen species (ROS), lipid peroxidation (LPO), protein carbonyl content, glutathione (GSH) content, mitochondrial function, mitochondrial membrane potential (MMP) and mitochondrial swelling were assessed. Our results showed that ROS and LPO at 100μM and protein carbonylation at 600μM concentrations of TR were significantly increased. GSH was decreased specifically at 600μM concentration. Mitochondrial function, MMP and mitochondrial swelling decreased in isolated rat liver mitochondria after exposure to 100 and 300μM, respectively. This study suggested that TR at therapeutic and toxic levels by single exposure could not induce mitochondrial toxicity. But, in lethal concentration (≥100μM), TR induced oxidative damage and mitochondria dysfunction. This study suggested that ROS overproduction by increasing of TR concentration induced mitochondrial dysfunction and caused mitochondrial damage via Complex II and membrane permeability transition pores disorders, MMP collapse and mitochondria swelling.