Respiration Of Rat Liver Mitochondria Research Articles

The stimulation of succinate-fueled respiration of rat liver mitochondria in state 4 by α,ω-hexadecanedioic acid without induction of proton conductivity of the inner membrane. Intrinsic uncoupling of the bc1 complex

The paper shows that natural α,ω-dioic acid, α,ω-hexadecanedioic acid (HDA), is able to stimulate the respiration of succinate-fueled rat liver mitochondria in state 4 without induction of proton conductivity of the inner membrane. This effect of HDA is less pronounced in glutamate/malate-fueled mitochondria, as well as in the case of ascorbate/TMPD or ascorbate/ferrocyanide substrate systems, which transfer electrons directly to cytochrome c. It is noted that HDA-induced stimulation of respiration is not associated with damage to the inner membrane in a part of mitochondria and with shunting of electrons through the bc1 complex. Therefore, HDA can be considered as a natural decoupling agent. Specific inhibitors of the bc1 complex (antimycin A and myxothiazole) as well as malonate and dithionitrobenzoate were used in the inhibitory analysis. These and other experiments have shown that during the oxidation of succinate in liver mitochondria, the decoupling effect of HDA is mainly carried out at the level of the bc1 complex. We hypothesized that HDA is capable of promoting the cyclic transport of protons within the bc1 complex and thus switch this complex to the idle mode of operation (intrinsic uncoupling of the bc1 complex). Induction of free respiration in liver mitochondria by HDA at the level of the bc1 complex is considered as one of the "rescue pathways" of hepatocytes in various pathological conditions, accompanied by disorders of carbohydrate and lipid metabolism and increased oxidative stress.

Itaconic acid impairs the mitochondrial function by the inhibition of complexes II and IV and induction of the permeability transition pore opening in rat liver mitochondria

Itaconic acid (methylene-succinic acid, ItA) is an unsaturated dicarboxylic acid that is secreted by mammalian macrophages in response to a pro-inflammatory stimulus and shows an anti-inflammatory/antibacterial effect. Being a mitochondrial metabolite, it exhibits an inhibitory activity on succinate dehydrogenase and subsequently induces mitochondrial dysfunction. The present study has shown that ItA dose-dependently inhibited ADP- and DNP-stimulated (uncoupled) respiration of rat liver mitochondria energized with succinate. This effect of ItA could be related to the suppression of the activity of complex II and the combined activity of complexes II + III of the respiratory chain. At the same time, ItA had no effect on the activity of the dicarboxylate carrier, which catalyzes the transport of succinate across the inner mitochondrial membrane. It was found that 4 mM ItA diminished the rates of ADP- and DNP-stimulated mitochondrial respiration supported by the substrates of complex I glutamate and malate. A study of the effect of ItA on the activity of complexes of the respiratory chain showed that it decreases the activity of complex IV. It was observed that 4 mM ItA inhibited the rate of H2O2 production by mitochondria. At the same time, ItA promoted the opening of the cyclosporin A-sensitive Ca2+-dependent permeability transition pore. The latter was revealed as the decrease in the calcium retention capacity of mitochondria and the stimulation of release of cytochrome c from the organelles. ItA by itself promoted the cytochrome c release from mitochondria. Possible mechanisms of the effect of ItA on mitochondrial function are discussed.

A neuroglobin-based high-affinity ligand trap reverses carbon monoxide–induced mitochondrial poisoning

Carbon monoxide (CO) remains the most common cause of human poisoning. The consequences of CO poisoning include cardiac dysfunction, brain injury, and death. CO causes toxicity by binding to hemoglobin and by inhibiting mitochondrial cytochrome c oxidase (CcO), thereby decreasing oxygen delivery and inhibiting oxidative phosphorylation. We have recently developed a CO antidote based on human neuroglobin (Ngb-H64Q-CCC). This molecule enhances clearance of CO from red blood cells in vitro and in vivo Herein, we tested whether Ngb-H64Q-CCC can also scavenge CO from CcO and attenuate CO-induced inhibition of mitochondrial respiration. Heart tissue from mice exposed to 3% CO exhibited a 42 ± 19% reduction in tissue respiration rate and a 33 ± 38% reduction in CcO activity compared with unexposed mice. Intravenous infusion of Ngb-H64Q-CCC restored respiration rates to that of control mice correlating with higher electron transport chain CcO activity in Ngb-H64Q-CCC-treated compared with PBS-treated, CO-poisoned mice. Further, using a Clark-type oxygen electrode, we measured isolated rat liver mitochondrial respiration in the presence and absence of saturating solutions of CO (160 μm) and nitric oxide (100 μm). Both CO and NO inhibited respiration, and treatment with Ngb-H64Q-CCC (100 and 50 μm, respectively) significantly reversed this inhibition. These results suggest that Ngb-H64Q-CCC mitigates CO toxicity by scavenging CO from carboxyhemoglobin, improving systemic oxygen delivery and reversing the inhibitory effects of CO on mitochondria. We conclude that Ngb-H64Q-CCC or other CO scavengers demonstrate potential as antidotes that reverse the clinical and molecular effects of CO poisoning.

3,5-Bis(3-dimethylaminomethyl-4-hydroxybenzylidene)-4-piperidone and related compounds induce glutathione oxidation and mitochondria-mediated cell death in HCT-116 colon cancer cells

This study aims at investigating the cytotoxicity and some of the modes of action of 3,5-bis(3-dimethylamino-4-hydroxybenzylidene)-4-piperidone trihydrochloride 3 and two related compounds 2 (which lacks the dimethylaminomethyl groups) and 4 (which has an additional dimethylaminoethyl substituent in both aryl rings) in order to ascertain the contribution of dimethylaminoethyl substituent to bioactivity. The bioactivities of 2–4 were compared with curcumin 5. Both 2 and 3 displayed submicromolar GI50 values towards HCT-116 cells and were significantly more potent than 4, 5 and 5-fluorouracil (5-FU). All of the compounds displayed greater toxicity towards HCT-116 cells than human CRL-1790 non-malignant colon cells. In HCT-116 cells, the compounds 2, 3 and 5 increased the ratio of oxidised to reduced glutathione and destabilized the mitochondrial membrane potential. Both 2 and 5 produced an increase in mitochondrial superoxide and a burst in intracellular reactive oxygen species in HCT 116 cells. In addition, 2 and 4 stimulated respiration in rat liver mitochondria while 2 and 5 induced mitochondrial swelling. The results suggest that 2 and 5 cause oxidation or cross-linking of the thiols which control the mitochondrial permeability transition.

Effects of manganese on potassium outflow from erythrocytes and on respiration of rat liver mitochondria

In this work, effects of manganese on respiration of rat liver mitochondria and the rate of K+ outflow from rat erythrocytes are studied in a broad range of concentrations. It is shown that manganese ions at low concentrations (1 × 10–7–3 × 10–5 М) inhibit K+ outflow from rat erythrocytes; this can be used to prevent their lysis. At high concentrations (1 × 10–4–1 × 10–3 M), manganese activates K+ outflow from the erythrocytes but inhibits the valinomycin-induced outflow of the ion from the erythrocytes. This fact is an indication of manganese influence on physicochemical properties of membranes. At low concentrations manganese does not affect parameters of respiration and oxidative phosphorylation of rat liver mitochondria, while at high concentrations it exerts acceleration of the mitochondrial respiration, i.e., uncouples respiration from phosphorilation and, hence, inhibits ATP synthesis.

Aluminum Phosphide Poisoning in Animals

Several articles have showed the effect of ALP toxicity on different organs. Toxicity mechanisms are not clearly understood yet. Due to the potential increased use of ALP as a fumigant and the lack of adequate toxicity data, previous studies were re-evaluated to characterize the epidemiological, toxicological, and clinical/ pathological aspects of ALP poisoning and its management. Related terms were looked up in bibliographical databases such as the Tehran University Medical Science Digital Library, PubMed, Scopus, Google Scholar, and British library. The studies suggest that phosphine targets the mitochondria and inhibits respiration in rat liver mitochondria, insect mitochondria, and intact nematodes. On the other hand, glutathione (GSH) levels are reduced in various tissues of ALP-poisoned rats, while remaining unchanged in insects and mammalian cells. Also, acetylcholine signaling is an important component of phosphine toxicity. Phosphine (PH3) induces oxidative stress and lipid peroxidation in insects, mammalian cells, and other animals. There is no known antidote for ALP intoxication; but, melatonin as an effective antidote protects against oxidative damage in the brain, lung, and liver of the rats and suggests the involvement of ROS in the genotoxicity of PH3. Cholinesterase inhibition responds to treatment with atropine, pralidoxime, and oral sweet almond oil, especially if used immediately after ALP poisoning. Several treatments have been used in animals some of which have not been tested in human-beings yet. Such treatments should be given in controlled situations with the hope they may be helpful in treating these patients.

Tl+ Showed Negligible Interaction with Inner Membrane Sulfhydryl Groups of Rat Liver Mitochondria, but Formed Complexes with Matrix Proteins

The effects of Tl(+) on protein sulfhydryl (SH) groups, swelling, and respiration of rat liver mitochondria (RLM) were studied in a medium containing TlNO3 and sucrose, or TlNO3 and KNO3 as well as glutamate plus malate, or succinate plus rotenone. Detected with Ellman's reagent, an increase in the content of the SH groups was found in the inner membrane fraction, and a simultaneous decline was found in the content of the matrix-soluble fraction for RLM, incubated and frozen in 25-75 mM TlNO3 . This increase was greater in the medium containing KNO3 regardless of the presence of Ca(2+) . It was eliminated completely for RLM injected in the medium containing TlNO3 and then washed and frozen in the medium containing KNO3 . Calcium-loaded RLM showed increased swelling and decreased respiration. These results suggest that a ligand interaction of Tl(+) with protein SH groups, regardless of the presence of calcium, may underlie the mechanism of thallium toxicity.

Structure–activity relationships for perfluoroalkane-induced in vitro interference with rat liver mitochondrial respiration

Perfluorinated alkyl acids (PFAAs) represent a broad class of commercial products designed primarily for the coatings industry. However, detection of residues globally in a variety of species led to the discontinuation of production in the U.S. Although PFAAs cause activation of the PPARα and CAR nuclear receptors, interference with mitochondrial bioenergetics has been implicated as an alternative mechanism of cytotoxicity. Although the mechanisms by which the eight carbon chain PFAAs interfere with mitochondrial bioenergetics are fairly well described, the activities of the more highly substituted or shorter chain PFAAs are far less well characterized. The current investigation was designed to explore structure–activity relationships by which PFAAs interfere with mitochondrial respiration in vitro. Freshly isolated rat liver mitochondria were incubated with one of 16 different PFAAs, including perfluorinated carboxylic, acetic, and sulfonic acids, sulfonamides and sulfamido acetates, and alcohols. The effect on mitochondrial respiration was measured at five concentrations and dose–response curves were generated to describe the effects on state 3 and 4 respiration and respiratory control. With the exception of PFOS, all PFAAs at sufficiently high concentrations (>20μM) stimulated state 4 and inhibited state 3 respiration. Stimulation of state 4 respiration was most pronounced for the carboxylic acids and the sulfonamides, which supports prior evidence that the perfluorinated carboxylic and acetic acids induce the mitochondrial permeability transition, whereas the sulfonamides are protonophoric uncouplers of oxidative phosphorylation. In both cases, potency increased with increasing carbon number, with a prominent inflection point between the six and eight carbon congeners. The results provide a foundation for classifying PFAAs according to specific modes of mitochondrial activity and, in combination with toxicokinetic considerations, establishing structure–activity-based boundaries for initial estimates of risk for noncancer endpoints for PFAAs for which minimal in vivo toxicity testing currently exists.

Toxicity assessments of nonsteroidal anti-inflammatory drugs in isolated mitochondria, rat hepatocytes, and zebrafish show good concordance across chemical classes

To reduce costly late-stage compound attrition, there has been an increased focus on assessing compounds in in vitro assays that predict attributes of human safety liabilities, before preclinical in vivo studies are done. Relevant questions when choosing a panel of assays for predicting toxicity are (a) whether there is general concordance in the data among the assays, and (b) whether, in a retrospective analysis, the rank order of toxicity of compounds in the assays correlates with the known safety profile of the drugs in humans. The aim of our study was to answer these questions using nonsteroidal anti-inflammatory drugs (NSAIDs) as a test set since NSAIDs are generally associated with gastrointestinal injury, hepatotoxicity, and/or cardiovascular risk, with mitochondrial impairment and endoplasmic reticulum stress being possible contributing factors. Eleven NSAIDs, flufenamic acid, tolfenamic acid, mefenamic acid, diclofenac, meloxicam, sudoxicam, piroxicam, diflunisal, acetylsalicylic acid, nimesulide, and sulindac (and its two metabolites, sulindac sulfide and sulindac sulfone), were tested for their effects on (a) the respiration of rat liver mitochondria, (b) a panel of mechanistic endpoints in rat hepatocytes, and (c) the viability and organ morphology of zebrafish. We show good concordance for distinguishing among/between NSAID chemical classes in the observations among the three approaches. Furthermore, the assays were complementary and able to correctly identify “toxic” and “non-toxic” drugs in accordance with their human safety profile, with emphasis on hepatic and gastrointestinal safety. We recommend implementing our multi-assay approach in the drug discovery process to reduce compound attrition.

Electrochemical determination of hydrogen peroxide production by isolated mitochondria: A novel nanocomposite carbon–maghemite nanoparticle electrode

Abstract A simple carbon paste electrode modified with novel maghemite (γ-Fe 2 O 3 ) nanoparticles, characterized by a mean diameter of about 10 nm and specific surface properties (called surface active maghemite nanoparticles, SAMNs) has been developed. The electrode is able to catalyze the electro-reduction of hydrogen peroxide at low applied potentials (−0.1 V vs. SCE). In order to improve the electrocatalytic properties of the modified electrode and to protect electrode surface from fouling due to the presence of biological materials, a surfactant, namely cetyltrimethylammonium bromide, was introduced, as monomolecular layer, on electrode surface. At −0.1 V, the sensitivity of the modified nanocomposite electrode was 58 nA μM −1 cm −2 , with a detection limit of 2.78 μM, in the 0.01–1.5 mM H 2 O 2 concentration range, revealing to be one of the most sensitive electrodes described in literature. The resulting electrode was used to determine native monoamine oxidase activity on intact mitochondria suspensions, allowing the determination of catalytic parameters of the enzyme. Following this new electrochemical methodology, we implemented a novel electrochemical assay for the investigation of aerobic metabolism in preparations of isolated mitochondria revealing active H 2 O 2 production by respiring rat liver mitochondria. The present work demonstrates the feasibility of these novel maghemite nanoparticles modified electrode as efficient hydrogen peroxide electro-catalyst, for the application on biological samples at low applied potentials.

T-2307 Causes Collapse of Mitochondrial Membrane Potential in Yeast

T-2307, an arylamidine compound, has been previously reported to have broad-spectrum in vitro and in vivo antifungal activities against clinically significant pathogens, including Candida species, Cryptococcus neoformans, and Aspergillus species, and is now undergoing clinical trials. Here we investigated the mechanism of action of T-2307 using yeast cells and mitochondria isolated from yeast and rat liver. Nonfermentative growth of Candida albicans and Saccharomyces cerevisiae in glycerol medium, in which yeasts relied on mitochondrial respiratory function, was inhibited at 0.001 to 0.002 μg/ml (0.002 to 0.004 μM) of T-2307. However, fermentative growth in dextrose medium was not inhibited by T-2307. Microscopic examination using Mitotracker fluorescent dye, a cell-permeant mitochondrion-specific probe, demonstrated that T-2307 impaired the mitochondrial function of C. albicans and S. cerevisiae at concentrations near the MIC in glycerol medium. T-2307 collapsed the mitochondrial membrane potential in mitochondria isolated from S. cerevisiae at 20 μM. On the other hand, in isolated rat liver mitochondria, T-2307 did not have any effect on the mitochondrial membrane potential at 10 mM. Moreover, T-2307 had little inhibitory and stimulatory effect on mitochondrial respiration in rat liver mitochondria. In conclusion, T-2307 selectively disrupted yeast mitochondrial function, and it was also demonstrated that the fungal mitochondrion is an attractive antifungal target.

Derivatives of Rhodamine 19 as Mild Mitochondria-targeted Cationic Uncouplers

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C(12)R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H(+) ions was generated in the presence of C(12)R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C(12)R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C(12)R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C(12)R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease.

Alkylrhodamines as Cationic Protonophores

Mild uncoupling of mitochondria assumes that partial decrease in membrane potential is beneficial for cells especially under some pathological conditions, suggesting that uncouplers are good candidates for therapeutic uses. The majority of known protonophoric uncouplers are weak acids capable to permeate across membranes in neutral and anionic forms. In the present study, protonophoric activity of a series of derivatives of cationic rhodamine 19 (including dodecylrhodamine C12R1 and its conjugate with plastoquinone SkQR1) but not of similar derivatives of rhodamine B was revealed by using a variety of assays. In planar bilayer lipid membranes, diffusion potential of hydrogen ions was formed in the presence of C12R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine similarly to the well-known uncoupler FCCP. C12R1 and SkQR1 also stimulated respiration of rat liver mitochondria and decreased their membrane potential as measured by DiS-C3-5. The protonophoric activity of alkylrhodamines strongly depended on the number of carbon atoms in their alkyl chain (n): it was maximal at n about 10-12 and substantially decreased at n = 16. We surmise that the proton transport involves translocation of neutral and protonated cationic forms of C12R1 across a membrane. Based on the fact that SkQR1 accumulates in mitochondria of mammalian cells and protects them from oxidative stress (Bakeeva et al. 2008, Biochemistry Moscow, 73:1288-1299), it can be proposed that the protonophore-induced uncoupling could be involved in the protective effect of SkQR1 along with the direct antioxidant effect of plastoquinone.

Hydro‐ethanolic extract of cashew tree (Anacardium occidentale) nut and its principal compound, anacardic acid, stimulate glucose uptake in C2C12 muscle cells

Products of cashew tree (Anacardium occidentale) are used in traditional medicine for various ailments, including diabetes. The anti-diabetic properties of cashew plant parts were studied using differentiated C2C12 myoblasts (myotubes) and rat liver mitochondria. Hydroethanolic extract of cashew seed (CSE) and its active component, anacardic acid (AA), stimulated glucose transport into C2C12 myotubes in a concentration-dependent manner. Extracts of other parts (leaves, bark and apple) of cashew plant were inactive. Significant synergistic effect on glucose uptake with insulin was noticed at 100 μg/mL CSE. CSE and AA caused activation of adenosine monophosphate-activated protein kinase in C2C12 myotubes after 6 h of incubation. No significant effect was noticed on Akt and insulin receptor phosphorylation. Both CSE and AA exerted significant uncoupling of succinate-stimulated respiration in rat liver mitochondria. Activation of adenosine monophosphate-activated protein kinase by CSE and AA likely increases plasma membrane glucose transporters, resulting in elevated glucose uptake. In addition, the dysfunction of mitochondrial oxidative phosphorylation may enhance glycolysis and contribute to increased glucose uptake. These results collectively suggest that CSE may be a potential anti-diabetic nutraceutical.

The centenary of medical thoracoscopy

<i>Background</i>—The “topical” effect of non-steroidal anti-inflammatory drugs (NSAIDs) seems to be an important cause of NSAID induced gastrointestinal damage. <i>Aim</i>—To examine the possible mechanism of the “topical” phase of damage in the small intestine. <i>Methods</i>—Electron microscopy and subcellular organelle marker enzyme studies were done in rat small intestine after oral administration of indomethacin (doses varied between 5 and 30 mg/kg). The effect of conventional and non-acidic NSAIDs on rat liver mitochondrial respiration was measured in vitro in a Clarke-type oxygen electrode. <i>Results</i>—The subcellular organelle marker enzymes showed mitochondrial and brush border involvement within an hour of indomethacin administration. Electron microscopy showed dose dependent mitochondrial changes following indomethacin administration consistent with uncoupling of oxidative phosphorylation (or inhibition of electron transport) which were indistinguishable from those seen with the uncoupler dinitrophenol. Parenteral indomethacin caused similar changes, but not in rats with ligated bile ducts. A range of NSAIDs, but not paracetamol or non-acidic NSAIDs which have a favourable gastrointestinal tolerability profile, uncoupled oxidative phosphorylation in vitro at micromolar concentrations and inhibited respiration at higher concentrations. In vivo studies with nabumetone and aspirin further suggested that uncoupling or inhibition of electron transport underlies the “topical” phase of NSAID induced damage. <i>Conclusion</i>—Collectively, these studies suggest that NSAID induced changes in mitochondrial energy production may be an important component of the “topical” phase of damage induction.

The effect of 6-deoxyclitoriacetal from Clitoria macrophylla Wall. on rat liver mitochondrial respiration and ATPase activity

A Thai plant Clitoria macrophylla Wall. was claimed to possess some pharmacological activities. The phytochemical studies reported a rotenoid compound, 6-deoxyclitoriacetal, which exhibits the cytotoxic effect in several cell line experiments. This report was to study the effect of 6-deoxyclitoriacetal on rat liver mitochondria, which may associated to its cytotoxic phenomena. Mitochondrial suspension was prepared from male Wistar rat liver by standard method (Hogeboom, 1955). The mitochondrial functions were demonstrated by measuring the oxygen consumption by standard polarographic method. The ATPase activity was measured from the amount of inorganic phosphate released from ATP hydrolysis. Protein content was measured by the method of Lowry et. al. (1951) 6-Deoxyclitoriacetal inhibited the oxygen consumption and oxidative phosphorylation with NAD+-linked substrates (glutamate+malate, ?-ketoglutarate and ?-hydroxybutyrate) but not with succinate. The compound also inhibited the NADH oxidation in osmotic-shocked mitochondria. These suggested that 6-deoxyclitoriacetal act as a mitochondrial electron transport inhibitor by inhibiting the complex I. In addition, 6-deoxyclitoriacetal activated the mitochondrial ATPase and led to decrease mitochondria ATP synthesis. 6-Deoxyclitoiacetal, a new rotenoid isolated from Clitoria macrophylla Wall. demonstrated its inhibitory effect on site I mitochondrial respiratory chain and stimulate APTase activity which may partly explain its cytotoxic effect.

E-2-[3-(3,4-Dichlorophenyl)-1-oxo-2-propenyl]-3-methylquinoxaline-1,4-dioxide: A lead antitubercular agent which alters mitochondrial respiration in rat liver

A series of 2-(3-aryl-1-oxo-2-propenyl)-3-methylquinoxaline-1,4-dioxides 1a– l and 2-acetyl-3-methylquinoxaline-1,4-dioxide 2 were evaluated against Mycobacterium tuberculosis H 37Rv. With the exception of the 4-nitro analog 1k, significant antitubercular potencies were observed in series 1 and 2 which have IC 50 values in the range of 1–23 μM. Negative correlations were noted between the IC 50 values of 1a– j, l towards M. tuberculosis and both the σ and π constants of the substituents in the benzylidene aryl ring. In particular, 1h emerged as a lead compound having IC 50 and IC 90 figures of 1.03 μM and 1.53 μM, respectively. This molecule affected respiration in rat liver mitochondria which is likely one way that 1h and the bioactive analogs exert their antitubercular properties. The quinoxaline 2, which lacks an α,β-unsaturated group, has no effect on mitochondrial respiration using concentrations which inhibit the growth of M. tuberculosis.

Gender-dependence of hyperthermia-induced changes in respiration of rat liver mitochondria

Gender-dependence of hyperthermia-induced changes in respiration of rat liver mitochondria

Toxicity assessment of the herbicide metolachlor comparative effects on bacterial and mitochondrial model systems

Metolachlor is one of the most intensively used chloroacetamide herbicides. However, its effects on the environment and on non-target animals and humans as well as its interference at a cell/molecular level have not yet been fully elucidated. The aim of this study was: firstly, to evaluate the potential toxicity of metolachlor at a cell/subcellular level by using two in vitro biological model systems (a strain of Bacillus stearothermophilus and rat liver mitochondria); secondly, to evaluate the relative sensibility of these models to xenobiotics to reinforce their suitability for pollutant toxicity assessment. Our results show that metolachlor inhibits growth and impairs the respiratory activity of B. stearothermophilus at concentrations two to three orders of magnitude higher than those at which bacterial cells are affected by other pesticides. Also at concentrations significantly higher than those of other pesticides, metolachlor depressed the respiratory control ratio, membrane potential and respiration of rat liver mitochondria when malate/glutamate or succinate were used as respiratory substrates. Moreover, metolachlor impaired the respiratory activity of rat liver mitochondria in the same concentration range at which it inhibited bacterial respiratory system (0.4–5.0 μmol/mg of protein). In conclusion, the high concentration range at which metolachlor induces toxicity in vitro suggests that this compound is safer than other pesticides previously studied in our laboratory, using the same model systems. The good parallelism between metolachlor effects on both models and the toxicity data described in the literature, together with results obtained in our laboratory with other compounds, indicate the suitability of these systems to assess toxicity in vitro.

Acyl-CoA-induced generation of reactive oxygen species in mitochondrial preparations is due to the presence of peroxisomes

Preparations of rat liver mitochondria, but not of brain and heart mitochondria, produce large quantities of reactive oxygen species (ROS) in the presence of palmitoyl-CoA and other long-chain acyl-CoAs. Palmitoyl-CoA inhibited respiration of rat liver mitochondria with glutamate plus malate or with succinate as substrate. However, ROS production induced by acyl-CoA was independent of respiration inhibition, as it was also observed in antimycin A- and rotenone-inhibited mitochondria and in submitochondrial particles in the absence of respiratory substrates (other than acyl-CoA). Increased ROS production by acyl-CoA in rat liver mitochondrial preparations was observed when measured in the external medium using Amplex red as a probe, but not inside mitochondria using the internal fluorescent probe MitoSOX or aconitase activity as the “intrinsic” indicator of ROS generation in the matrix compartment. Stimulation by acyl-CoA of ROS generation was higher in “light” mitochondrial preparations that were enriched in peroxisomes, as assayed by urate oxidase. It is concluded that stimulation of ROS production in preparations of rat liver mitochondria could be ascribed to contaminating peroxisomes. Preparations of rat brain and heart mitochondria were not or were much less contaminated with peroxisomes, as indicated by low urate oxidase activity.