Isolated Rat Liver Research Articles

Pharmacological Insights into Salvia Rosmarinus: A Natural Remedy with Diverse Therapeutic Potential

Rosmarinus officinalis, commonly known as rosemary, is an aromatic herb originating from the Mediterranean region, primarily processed in Murcia, Southeast Spain. It is highly valued in the United States and Europe for its antioxidant properties. Rosemary extracts are renowned for their diverse therapeutic applications, such as liver protection, Alzheimer's disease treatment, and inhibiting the growth of new blood vessels. These extracts also play a vital role in food preservation by preventing oxidation and microbial contamination, offering a natural alternative to synthetic antioxidants. In medicinal use, rosemary helps treat conditions like renal colic, menstrual pain, respiratory disorders, and general pain relief. It aids digestion and bile flow due to its carminative and cholagogue properties and has diuretic effects that enhance urine production. Moreover, rosemary exhibits antiepileptic effects and may benefit fertility. The plant's biosynthetic pathways produce phenolic compounds, including caffeic acid (CA) and dihydroxy-phenyl-lactic acid (DOPL). Shikimic acid is crucial in synthesizing amino acids like phenylalanine, tyrosine, and tryptophan. The process involves converting phenylalanine into cinnamic acid, leading to the formation of Rosmarinus acid (RA), which is an ester of DOPL and has traditionally been used for its liver-protective and bile-stimulating properties, with Rosemary studies confirming its effectiveness in increasing bile flow and protecting against liver toxicity. Rosemary extracts also reduce the formation of malonaldehyde and the release of enzymes in isolated rat liver cells, confirming their hepatoprotective effects. Rosemary also holds promise in cancer prevention, as its dietary non-nutrients effectively inhibit carcinogenesis. RA, absorbed through oral and parenteral routes, has a half-life of 1.8 hours and is detectable in various tissues, especially in the lungs. The herb's antioxidants, including vitamins C and E, and beta carotene, help mitigate oxidative stress, a major factor in many diseases and aging. These antioxidants prevent DNA damage, protein harm, and uncontrolled lipid peroxidation, which are crucial in disease development.

Effects of 2,4-dichlorophenoxyacetic acid (2,4-D), isolated and in a formulated product, on the functional parameters of isolated rat liver mitochondria

2,4-Dichlorophenoxyacetic acid (2,4-D) is a widely used herbicide with known implications for environmental and biological systems. Previous studies indicate its potential to interact with cellular structures, potentially impacting cellular energy and metabolism. This study aimed to investigate the toxicological effects of this molecule on isolated rat liver mitochondria, evaluating both isolated and formulated 2,4-D to comprehend the differential impacts on mitochondrial function. Our investigations did not reveal significant induction of oxidative stress, mitochondrial swelling or impacts on mitochondrial respiration; however, exposure to both forms of 2,4-D affected mitochondrial membrane integrity and function in a concentration-dependent manner, notably impacting mitochondrial ATP levels and membrane potential. Comparatively, the formulated product showed toxicity at lower concentrations regarding interaction with membranes and ATP levels (from 0.4 μM on) and isolated 2,4-D showed toxicity at lower concentrations regarding membrane potential dissipation (from 10 μM on). Notably, the effects on ATP levels and membrane fluidity suggest interactions within the mitochondrial lipid bilayer. The findings highlight that excipients in the commercial formulation of 2,4-D potentially alter its effects, underlining the importance of evaluating not only active ingredients but also formulations.

Phytochemical analysis, antioxidant activity and modulatory effect of aqueous leaf extract of Pistacia lentiscus L on oxaliplatin-induced oxidative stress in isolated rat liver mitochondria

This study evaluates the antioxidant effects of aqueous leaf extract of Pistacia lentiscus (ALEPL) and its potential to counteract oxaliplatin (OXA)-induced mitochondrial oxidative stress in rat livers, a common side effect of chemotherapy in cancer treatment. Bioactive compounds were identified using High-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (HPLC-MS and MS), with Fourier-Transform Infrared Spectroscopy (FTIR) and Atomic Absorption Spectrophotometry (AAS) for chemical and mineral analysis. ALEPL showed notable antioxidant activity, with IC50 values of 4.30 ± 0.27 μg/mL for DPPH (2,2-Diphenyl-1-picrylhydrazyl) scavenging, 13.64 ± 0.51 μg/mL for reducing power, 32.62 ± 5.32 μg/mL for hydroxyl radical scavenging, and 205.08 ± 25.77 μg/mL for superoxide anion radical scavenging. In ex vivo experiments, mitochondria isolated from Wistar rat livers were treated with OXA and ALEPL in a dose-dependent manner. ALEPL pretreatment effectively restored mitochondrial antioxidant enzyme activities, increased glutathione (GSH) levels, and reduced lipid peroxidation (MDA) caused by OXA. These findings suggest that ALEPL has the potential to act as a natural antioxidant to support cancer treatment by mitigating chemotherapy-induced oxidative stress. Future studies could explore its application as an adjuvant in clinical settings to enhance the efficacy of chemotherapy while reducing its side effects.

Spontaneous reversal of small molecule-induced mitochondrial uncoupling: the case of anilinothiophenes.

Tissue specificity can render mitochondrial uncouplers more promising asleading compounds for creating drugs against serious diseases. In searchof tissue-specific uncouplers, we address anilinothiophenes as possible glutathione-S-transferase substrates (GST). Earlier, 'cyclic' uncoupling activity was reported for 5-bromo-N-(4-chlorophenyl)-3,4-dinitro-2-thiophenamine (BDCT) in isolated rat liver mitochondria (RLM). The mechanism by which BDCT induced two-phase changes in mitochondrial respiration (stimulation followed by deceleration) was unknown. To clarify this issue, we synthesized BDCT and its two analogues. Among these, 5-bromo-3,4-dinitro-N-(4-nitrophenyl)-2-thiophenamine (BDNT) appeared to be the most effective as a mitochondrial uncoupler, decreasing membrane potential and stimulating respiration at submicromolar concentrations. Importantly, BDNT exerted two-phase changes in both mitochondrial membrane potential and respiration rate of RLM, which were enhanced by the addition of glutathione (GSH) but inhibited by the compounds capable of GSH depleting, such as 1-chloro-2,4-dinitrobenzene (CDNB). By contrast, the phase of recoupling was not observed in rat heart mitochondria (RHM). Remarkably, BDNT elicited mitochondrial depolarization in primary human fibroblasts but not in cultured human liver (HepG2) cells. By detecting proton-selective electrical current through planar bilayer lipid membranes, we demonstrated the ability of BDCT and BDNT to transfer protons across membranes. BDNT proved to be an anionic protonophore with a pKa of 7.38. By using LC-MS and capillary electrophoresis, we directly showed the formation of BDNT conjugates with GSH upon incubation with RLM but not RHM. Therefore, we hypothesize that GST is involved in the disappearance of the anilinothiophene uncoupling activity in RLM, ensuring the tissue-specific behavior of the uncoupler.

Effects of Salicylic and Acetylsalicylic Acids in Mitochondrial and Erythrocyte Membranes

For further clarification of the mechanisms of pharmacological effects of salicylic and acetylsalicylic acids, the interactions of these acids with mitochondrial and erythrocyte membranes were studied and the role of calcium ions in the effects of salicylic and acetylsalicylic acids was examined. Salicylic acid and to a lesser extent acetylsalicylic acid at 0.5−2.0 mM concentration effectively inhibited the respiratory activity of isolated rat liver mitochondria, by uncoupling respiration and phosphorylation processes, induced depolarization of the mitochondrial membrane and potentiated Ca2+-stimulated formation of mitochondrial permeability transition pores in EGTA-free media. Cyclosporine A and ruthenium red partially inhibited the mitochondrial pore opening process induced by salicylic and acetylsalicylic acids both in the absence and presence of Ca2+ ions. Salicylic acid (180–360 µM) markedly accelerated proton-induced lysis of human erythrocytes (at pH 3.2) and caused hyperpolarization of erythrocyte membranes (at pH 5.5, but not at pH 7.4), probably as a result of proton transfer to the cytoplasm of the cell. Thus, salicylic and acetylsalicylic acids interact with mitochondrial and plasma membranes, act as effective proton/Ca2+ ionophores and stimulate the mitochondrial calcium uniporter.

A compact machine perfusion device for whole blood perfusion in isolated rat liver.

We established a compact machine perfusion system for whole blood perfusion of rat liver by making use of oxygenation filters as an artificial lung. Livers removed from rats were divided into Krebs-Henseleit (control), 50% blood (hemoglobin: 7g/dL), and whole blood (hemoglobin: 14g/dL) groups, then perfused (total perfusate volume: 25ml) with a small oxygenation filter at 37°C for 120min. Blood or perfusate was collected over time, and blood gas and blood cell were measured. In addition, bile volume and portal venous pressure measurements were taken. In all groups, the partial pressure of oxygen was controlled to approximately 400mmHg. Flow rates were maintained at approximately about 20-30ml/min according to liver size. Portal venous pressure was normal in the 50% blood and whole blood groups, while lower than the reference value in the Krebs-Henseleit group. Twice as much bile was produced in the 50% blood and whole blood groups relative with the Krebs-Henseleit group. We observed no differences in hemoglobin and red blood cell levels. Lactate levels were normal in the 50% blood and whole blood groups, but were elevated in the Krebs-Henseleit group. Our compact perfusion system using oxygenation filters was able to maintain rat liver function by perfusing a small amount of extracorporeal blood. This system is simple and stable, and may contribute to the future development of machine perfusion systems.

A novel cytoprotective organ perfusion platform for reconstructing homeostasis of DCD liver while alleviating IRI injury

AbstractPump is a vital component for expelling the perfusate in small animal isolated organ normothermic machine perfusion (NMP) systems whose flexible structure and rhythmic contraction play a crucial role in maintaining perfusion system homeostasis. However, the continuous extrusion forming with the rigid stationary shaft of the peristaltic pumps can damage cells, leading to metabolic disorders and eventual dysfunction of transplanted organs. Here, we developed a novel biomimetic blood‐gas system (BBGs) for preventing cell damage. This system mimics the cardiac cycle and features an adjustable inspiratory‐to‐expiratory (IE) ratio to mitigate acidosis caused by continuous oxygen inhalation. In our study, adipose stem cells (ADSCs) were cultured within the circulatory system for 10 min, 2, and 4 h. Compared to the peristaltic pump, the BBGs significantly reduced cell apoptosis and morphological injury while enhancing cell proliferation and adhesion. Additionally, when the supernatant from ADSCs was introduced to LPS‐induced macrophages for 24 h, the BBGs group demonstrated a more pronounced anti‐inflammatory effect, characterized by reduced M1 macrophage expression. Besides, with isolated rat livers from donation after circulatory death (DCD) perfusion with ADSCs for 6 h by the BBGs, we detected fewer apoptotic cells and a reduced inflammatory response, evidenced by down‐regulated TNF‐α expression. The development of BBGs demonstrates the feasibility of recreating physiological liquid–gas circulation in vitro, offering an alternative platform for isolated organ perfusion, especially for applications involving cell therapy.

Synthesis and comparative analysis of the cytotoxicity and mitochondrial effects of triphenylphosphonium and F16 maslinic and corosolic acid hybrid derivatives

The cytotoxic profile and antiproliferative and mitochondrial effects of triterpene acid conjugates with mitochondriotropic lipophilic triphenylphosphonium (TPP+) and F16 cations were evaluated. Maslinic and corosolic acids chosen as the investigation objects were synthesized from commercially available oleanolic and ursolic acids. Study of the cytotoxic activity of TPP+ and F16 triterpenoid derivatives against six tumor cell lines demonstrated a comparable synergistic effect in the anticancer activity, which was most pronounced in the case of MCF-7 mammary adenocarcinoma cells and Jurkat and THP-1 leukemia cells. The corosolic and maslinic acid hybrid derivatives caused changes in the progression of tumor cell cycle phases when present in much lower doses than their natural triterpene acid precursors. The treatment of tumor cell lines with the conjugates resulted in the cell cycle arrest in the G1 phase and increase in the cell population in the subG1 phase. The cationic derivatives of the acids were markedly superior to their precursors as inducers of hyperproduction of reactive oxygen species and more effectively decreased the mitochondrial potential in isolated rat liver mitochondria. We concluded that the observed cytotoxic effect of TPP+ and F16 triterpenoid conjugates is attributable to the ability of these compounds to initiate mitochondrial dysfunctions. Their cytotoxicity, antiproliferative action, and mitochondrial effects depend little on the type of cationic groups used.

SkQ3 Exhibits the Most Pronounced Antioxidant Effect on Isolated Rat Liver Mitochondria and Yeast Cells.

Oxidative stress is involved in a wide range of age-related diseases. A critical role has been proposed for mitochondrial oxidative stress in initiating or promoting these pathologies and the potential for mitochondria-targeted antioxidants to fight them, making their search and testing a very urgent task. In this study, the mitochondria-targeted antioxidants SkQ1, SkQ3 and MitoQ were examined as they affected isolated rat liver mitochondria and yeast cells, comparing SkQ3 with clinically tested SkQ1 and MitoQ. At low concentrations, all three substances stimulated the oxidation of respiratory substrates in state 4 respiration (no ADP addition); at higher concentrations, they inhibited the ADP-triggered state 3 respiration and the uncoupled state, depolarized the inner mitochondrial membrane, contributed to the opening of the mPTP (mitochondrial permeability transition pore), did not specifically affect ATP synthase, and had a pronounced antioxidant effect. SkQ3 was the most active antioxidant, not possessing, unlike SkQ1 or MitoQ, prooxidant activity with increasing concentrations. In yeast cells, all three substances reduced prooxidant-induced intracellular oxidative stress and cell death and prevented and reversed mitochondrial fragmentation, with SkQ3 being the most efficient. These data allow us to consider SkQ3 as a promising potential therapeutic agent to mitigate pathologies associated with oxidative stress.

Risk Assessment of Psychotropic Drugs on Mitochondrial Function Using In Vitro Assays.

Mitochondria are potential targets responsible for some drug- and xenobiotic-induced organ toxicities. However, molecular mechanisms of drug-induced mitochondrial toxicities are mostly unknown. Here, multiple in vitro assays were used to investigate the effects of 22 psychotropic drugs on mitochondrial function. The acute extracellular flux assay identified inhibitors of the electron transport chain (ETC), i.e., aripiprazole, phenytoin, and fluoxetine, an uncoupler (reserpine), substrate inhibitors (quetiapine, carbamazepine, buspirone, and tianeptine), and cytotoxic compounds (chlorpromazine and valproic acid) in HepG2 cells. Using permeabilized HepG2 cells revealed minimum effective concentrations of 66.3, 6730, 44.5, and 72.1 µM for the inhibition of complex-I-linked respiration for quetiapine, valproic acid, buspirone, and fluoxetine, respectively. Assessing complex-II-linked respiration in isolated rat liver mitochondria revealed haloperidol is an ETC inhibitor, chlorpromazine is an uncoupler in basal respiration and an ETC inhibitor under uncoupled respiration (IC50 = 135 µM), while olanzapine causes a mild dissipation of the membrane potential at 50 µM. This research elucidates some mechanisms of drug toxicity and provides some insight into their safety profile for clinical drug decisions.

The transient character of mitochondrial uncoupling by the popular fungicide fluazinam is specific for liver

The popular fungicide fluazinam is known to exhibit an unusual cyclic pattern of the protonophoric uncoupling activity in isolated rat liver mitochondria (RLM), with membrane deenergization followed by spontaneous recoupling in the minute scale, which is associated with glutathione conjugation of fluazinam catalyzed by glutathione-S-transferase (GST). Here, we compare the fluazinam effect on RLM with that on rat kidney (RKM) and heart (RHM) mitochondria by monitoring three bioenergetic parameters: oxygen consumption rate, mitochondrial membrane potential and reduction of nucleotides. Only in RLM, the uncoupling activity of fluazinam was transient, i.e. disappeared in a few minutes, whereas in RKM and RHM it was stable in this time scale. We attribute this difference to the increased activity of mitochondrial GST in liver. We report data on the detection of glutathione-fluazinam conjugates by mass-spectrometry, thin layer chromatography and capillary electrophoresis after incubation of fluazinam with RLM but not with RKM, which supports the assumption of the tissue specificity of the conjugation.

Hybrids of Sterically Hindered Phenols and Diaryl Ureas: Synthesis, Switch from Antioxidant Activity to ROS Generation and Induction of Apoptosis.

The utility of sterically hindered phenols (SHPs) in drug design is based on their chameleonic ability to switch from an antioxidant that can protect healthy tissues to highly cytotoxic species that can target tumor cells. This work explores the biological activity of a family of 45 new hybrid molecules that combine SHPs equipped with an activating phosphonate moiety at the benzylic position with additional urea/thiourea fragments. The target compounds were synthesized by reaction of iso(thio)cyanates with C-arylphosphorylated phenols containing pendant 2,6-diaminopyridine and 1,3-diaminobenzene moieties. The SHP/urea hybrids display cytotoxic activity against a number of tumor lines. Mechanistic studies confirm the paradoxical nature of these substances which combine pronounced antioxidant properties in radical trapping assays with increased reactive oxygen species generation in tumor cells. Moreover, the most cytotoxic compounds inhibited the process of glycolysis in SH-SY5Y cells and caused pronounced dissipation of the mitochondrial membrane of isolated rat liver mitochondria. Molecular docking of the most active compounds identified the activator allosteric center of pyruvate kinase M2 as one of the possible targets. For the most promising compounds, 11b and 17b, this combination of properties results in the ability to induce apoptosis in HuTu 80 cells along the intrinsic mitochondrial pathway. Cyclic voltammetry studies reveal complex redox behavior which can be simplified by addition of a large excess of acid that can protect some of the oxidizable groups by protonations. Interestingly, the re-reduction behavior of the oxidized species shows considerable variations, indicating different degrees of reversibility. Such reversibility (or quasi-reversibility) suggests that the shift of the phenol-quinone equilibrium toward the original phenol at the lower pH may be associated with lower cytotoxicity.

Efficiency of mitochondrial uncoupling by modified butyltriphenylphosphonium cations and fatty acids correlates with lipophilicity of cations: Protonophoric vs leakage mechanisms

In order to determine the share of protonophoric activity in the uncoupling action of lipophilic cations a number of analogues of butyltriphenylphosphonium with substitutions in phenyl rings (C4TPP-X) were studied on isolated rat liver mitochondria and model lipid membranes. An increase in the rate of respiration and a decrease in the membrane potential of isolated mitochondria were observed for all the studied cations, the efficiency of these processes was significantly enhanced in the presence of fatty acids and correlated with the octanol-water partition coefficient of the cations. The ability of C4TPP-X cations to induce proton transport across the lipid membrane of liposomes loaded with a pH-sensitive fluorescent dye increased also with their lipophilicity and depended on the presence of palmitic acid in the liposome membrane. Of all the cations, only butyl[tri(3,5-dimethylphenyl)]phosphonium (C4TPP-diMe) was able to induce proton transport by the mechanism of formation of a cation-fatty acid ion pair on planar bilayer lipid membranes and liposomes. The rate of oxygen consumption by mitochondria in the presence of C4TPP-diMe increased to the maximum values corresponding to conventional uncouplers; for all other cations the maximum uncoupling rates were significantly lower. We assume that the studied cations of the C4TPP-X series, with the exception of C4TPP-diMe at low concentrations, cause nonspecific leak of ions through lipid model and biological membranes which is significantly enhanced in the presence of fatty acids.

Conjugation of Triterpenic Acids of Ursane and Oleanane Types with Mitochondria-Targeting Cation F16 Synergistically Enhanced Their Cytotoxicity against Tumor Cells.

The present work shows the cytotoxic effects of novel conjugates of ursolic, oleanolic, maslinic, and corosolic acids with the penetrating cation F16 on cancer cells (lung adenocarcinoma A549 and H1299, breast cancer cell lines MCF-7 and BT474) and non-tumor human fibroblasts. It has been established that the conjugates have a significantly enhanced toxicity against tumor-derived cells compared to native acids and also demonstrate selectivity to some cancer cells. The toxic effect of the conjugates is shown to be due to ROS hyperproduction in cells, induced by the effect on mitochondria. The conjugates caused dysfunction of isolated rat liver mitochondria and, in particular, a decrease in the efficiency of oxidative phosphorylation, a decrease in the membrane potential, and also an overproduction of ROS by organelles. The paper discusses how the membranotropic- and mitochondria-targeted effects of the conjugates may be related to their toxic effects.

Modulators of mitochondrial ATP-sensitive potassium channel affect cytotoxicity of heavy metals: Action on isolated rat liver mitochondria and AS-30D ascites hepatoma cells

Heavy metals are ubiquitous environmental pollutants that are extremely dangerous for public health, but the molecular mechanisms of their cytotoxic action are still not fully understood. In the present work, the possible contribution of the mitochondrial ATP-sensitive potassium channel (mK(ATP)), which is usually considered protective for the cell, to hepatotoxicity caused by heavy metals was investigated using polarography and swelling techniques as well as flow cytometry. Using isolated liver mitochondria from adult male Wistar rats and various potassium media containing or not containing penetrating anions (KNO3, KSCN, KAcet, KCl), we studied the effect of mK(ATP) modulators, namely its blockers (5-hydroxydecanoate, glibenclamide, ATP, ADP) and activators (diazoxide, malonate), on respiration and/or membrane permeability in the presence of hepatotoxins such as Cd2+, Hg2+, and Cu2+. It has been shown for the first time that, contrary to Hg2+ and depending on media used, the mK(ATP) modulators affect Cd2+- and/or Cu2+-induced alterations in mitochondrial swelling and respiration rates, although differently, nevertheless, in the ways compatible with mK(ATP) participation in both these cases. On rat AS-30D ascites hepatoma cells, it was found that, unlike Cd2+, an increase in the production of reactive oxygen species was observed with the simultaneous use of Cu2+ and diazoxide; in addition, there was no protective effect of diazoxide against cell death, which also occurred in the presence of Cu2+. In conclusion, the relationships (functional, structural and/or regulatory) between mK(ATP), components of the mitochondrial electron transport chain (CI, CII-CIII and/or ATP synthase, CV) and mitochondrial permeability transition pores were discussed, as well as the role of these molecular structures in the mechanisms of the cytotoxic action of heavy metals.

Rosemary (Rosmarinus officinalis L.) Glycolic Extract Protects Liver Mitochondria from Oxidative Damage and Prevents Acetaminophen-Induced Hepatotoxicity.

Rosmarinus officinalis L. (rosemary) is an aromatic culinary herb. Native to the Mediterranean region, it is currently cultivated worldwide. In addition to its use as a condiment in food preparation and in teas, rosemary has been widely employed in folk medicine and cosmetics. Several beneficial effects have been described for rosemary, including antimicrobial and antioxidant activities. Here, we investigated the mechanisms accounting for the antioxidant activity of the glycolic extract of R. officinalis (Ro) in isolated rat liver mitochondria (RLM) under oxidative stress conditions. We also investigated its protective effect against acetaminophen-induced hepatotoxicity in vivo. A crude extract was obtained by fractionated percolation, using propylene glycol as a solvent due to its polarity and cosmeceutical compatibility. The quantification of substances with recognized antioxidant action revealed the presence of phenols and flavonoids. Dereplication studies carried out through LC-MS/MS and GC-MS, supported by The Global Natural Product Social Molecular Networking (GNPS) platform, annotated several phenolic compounds, confirming the previous observation. In accordance, Ro decreased the production of reactive oxygen species (ROS) elicited by Fe2+ or t-BOOH and inhibited the lipid peroxidation of mitochondrial membranes in a concentration-dependent manner in RLM. Such an effect was also observed in liposomes as membrane models. Ro also prevented the oxidation of mitochondrial protein thiol groups and reduced glutathione (GSH). In model systems, Ro exhibited a potent scavenger activity toward 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radicals and superoxide anions. It also demonstrated an Fe2+ chelating activity. Moreover, Ro did not exhibit cytotoxicity or dissipate the mitochondrial membrane potential (∆Ψ) in rat liver fibroblasts (BRL3A cells). To evaluate whether such antioxidant protective activity observed in vitro could also be achieved in vivo, a well-established model of hepatotoxicity induced by acute exposure to acetaminophen (AAP) was used. This model depletes GSH and promotes oxidative-stress-mediated tissue damage. The treatment of rats with 0.05% Ro, administered intraperitoneally for four days, resulted in inhibition of AAP-induced lipid peroxidation of the liver and the prevention of hepatotoxicity, maintaining alanine and aspartate aminotransferase (ALT/AST) levels equal to those of the normal, non-treated rats. Together, these findings highlight the potent antioxidant activity of rosemary, which is able to protect mitochondria from oxidative damage in vitro, and effects such as the antioxidant and hepatoprotective effects observed in vivo.

Pyridoxine 4′-α- and 5′-α-D-Glucosides are Taken Up and Metabolized by Isolated Rat Liver Cells

Uptake and metabolism of pyridoxine-4′-α-D-glucoside and pyridoxine-5′-α-D-glucoside by freshly isolated rat liver cells were studied using the membrane filtration method. The presence of the pyridoxine-α-glucosides at either 0.5 or 5.0 µmol/L had no inhibitory effect on the uptake of pyridoxine (0.5 µmol/L) by hepatocytes. The amount of pyridoxine-α-glucoside transported into the cells was only 10–12% of that of pyridoxine. The initial rates of uptake of the pyridoxine-α-glucosides were linear with concentrations up to 50 µmol/L. On entry into the cells, 78% of pyridoxine-5′-α-glucoside and 15% of the pyridoxine-4′-α-glucoside were hydrolyzed at the end of 60 min. Because pyridoxine-5′-α-glucoside is hydrolyzed readily and does not interfere with the transport of pyridoxine, the glucoside, if present in animal- or plant-derived foods, may be available as a source of vitamin B-6.

The flavonoids fisetin, apigenin, kaempferol, naringenin, naringin regulate respiratory activity and membrane potential of rat liver mitochondria and inhibit oxidative processes in erythrocytes.

Flavonoids, secondary plant metabolites, represent the most abundant heterogeneous group of phytochemicals. The aim of this study to compare antioxidant activity and regulatory properties of several representatives of different classes of flavonoids, fisetin, apigenin, kaempferol, naringenin, naringin, using liver mitochondria and erythrocytes as research objects. In the concentration range of 2.5-25 μM fisetin, apigenin, kaempferol, naringenin, and naringin dose-dependently prevented oxidative damage of erythrocytes induced by 700 μM tert-butyl hydroperoxide: accumulation of lipid peroxidation (LPO) products and oxidation of glutathione GSH. The IC50 values corresponding to the flavonoid concentration inhibiting the LPO process in erythrocyte membranes by 50%, were 3.9±0.8 μM in the case of fisetin, 6.5±1.6 μM in the case of kaempferol, 8.1±2.1 μM in the case of apigenin, 37.8±4.4 μM in the case of naringenin, and 64.7±8.6 μM in the case of naringin. The antioxidant effect of flavonoids was significantly higher in the membrane structures compared to the cytoplasm of cells. All flavonoids studied (10-50 μM) effectively inhibited the respiratory activity of isolated rat liver mitochondria and, with the exception of kaempferol, stimulated Ca²⁺-induced dissipation of the mitochondrial membrane potential. Cyclosporine A and ruthenium red inhibited flavonoid-stimulated Ca²⁺-dependent membrane depolarization, thus indicating that the mitochondrial calcium uniporter and the mitochondrial permeability transition pore opening were involved in the flavonoid effects. Flavonoids, as the redox-active compounds with antioxidant properties, are able to regulate mitochondrial potential and respiratory activity, and prevent mitochondrial oxidative stress. They can be considered as effective pharmacological agents or nutraceuticals.

Toluidine blue O directly and photodynamically impairs the bioenergetics of liver mitochondria: a potential mechanism of hepatotoxicity.

Toluidine blue O (TBO) is a phenothiazine dye that, due to its photochemical characteristics and high affinity for biomembranes, has been revealed as a new photosensitizer (PS) option for antimicrobial photodynamic therapy (PDT). This points to a possible association with membranous organelles like mitochondrion. Therefore, here we investigated its effects on mitochondrial bioenergetic functions both in the dark and under photostimulation. Two experimental systems were utilized: (a) isolated rat liver mitochondria and (b) isolated perfused rat liver. Our data revealed that, independently of photostimulation, TBO presented affinity for mitochondria. Under photostimulation, TBO increased the protein carbonylation and lipid peroxidation levels (up to 109.40 and 119.87%, respectively) and decreased the reduced glutathione levels (59.72%) in mitochondria. TBO also uncoupled oxidative phosphorylation and photoinactivated the respiratory chain complexes I, II, and IV, as well as the FoF1-ATP synthase complex. Without photostimulation, TBO caused uncoupling of oxidative phosphorylation and loss of inner mitochondrial membrane integrity and inhibited very strongly succinate oxidase activity. TBO's uncoupling effect was clearly seen in intact livers where it stimulated oxygen consumption at concentrations of 20 and 40μM. Additionally, TBO (40μM) reduced cellular ATP levels (52.46%) and ATP/ADP (45.98%) and ATP/AMP (74.17%) ratios. Consequently, TBO inhibited gluconeogenesis and ureagenesis whereas it stimulated glycogenolysis and glycolysis. In conclusion, we have revealed for the first time that the efficiency of TBO as a PS may be linked to its ability to photodynamically inhibit oxidative phosphorylation. In contrast, TBO is harmful to mitochondrial energy metabolism even without photostimulation, which may lead to adverse effects when used in PDT.

Antibiotic Pyrrolomycin as an Efficient Mitochondrial Uncoupler.

Pyrrolomycins C (Pyr_C) and D (Pyr_D) are antibiotics produced by Actinosporangium and Streptomyces. The mechanism of their antimicrobial activity consists in depolarization of bacterial membrane, leading to the suppression of bacterial bioenergetics through the uncoupling of oxidative phosphorylation, which is based on the protonophore action of these antibiotics [Valderrama et al., Antimicrob. Agents Chemother. (2019) 63, e01450]. Here, we studied the effect of pyrrolomycins on the isolated rat liver mitochondria. Pyr_C was found to be more active than Pyr_D and uncoupled mitochondria in the submicromolar concentration range, which was observed as the mitochondrial membrane depolarization and stimulation of mitochondrial respiration. In the case of mitoplasts (isolated mitochondria with impaired outer membrane integrity), the difference in the action of Pyr_C and Pyr_D was significantly less pronounced. By contrast, in inverted submitochondrial particles (SMPs), Pyr_D was more active as an uncoupler, which caused collapse of the membrane potential even at the nanomolar concentrations. The same ratio of the protonophoric activity of Pyr_D and Pyr_C was obtained by us on liposomes loaded with the pH indicator pyranine. The protonophore activity of Pyr_D in the planar bilayer lipid membranes (BLMs) was maximal at ~pH 9, i.e., at pH values close to pKa of this compound. Pyr_D functions as a typical anionic protonophore; its activity in the BLM could be reduced by the addition of the dipole modifier phloretin. The difference between the protonophore activity of Pyr_C and Pyr_D in the mitochondria and BLMs can be attributed to a higher ability of Pyr_C to penetrate the outer mitochondrial membrane.