Effects of 1,2-dibromoethane on glutathione metabolism in rat liver and kidney

There are few reports of the effects of glutathione-depleting agents administered for periods longer than 24 hr on the turnover of glutathione (GSH) in mammalian tissues. Studies of such effects are important in relation to the protection of tissues from damage from, for example, reactive metabolites derived from xenobiotics. In the investigation described here, 1,2-dibromoethane dibromide)-a widely used insecticide, nematocide, fungicide and petrol additive, which is hepato- and nephrotoxic-was administered to rats and the effects on non-protein thiol contents and GSH-related enzyme activities were determined in liver and kidney. The classical GSH-depleter diethylmaleate was used in parallel studies for comparative purposes.     

Deuterium isotope effect on the metabolism and toxicity of 1,2-dibromoethane

The metabolism, hepatotoxicity, and hepatic DNA damage of 1,2-dibromoethane (EDB) and tetradeutero-1,2-dibromoethane (d₄EDB) were compared in male Swiss-Webster mice. In vitro studies that measured bromide ion released from the substrate to monitor the rate of metabolism showed that the hepatic microsomal metabolism of EDB was significantly reduced by deuterium substitution, while metabolism by the hepatic glutathione S-transferases was unaffected. Three hours after ip administration of EDB or d₄EDB (50 mg/kg), there was 42% less bromide in the plasma of d₄EDB-treated mice than in the plasm of EDB-treated mice. This difference demonstrates a significant deuterium isotope effect on the metabolism of EDB in vivo. Although the metabolism of d₄EDB was less than that of EDB 3 hr after exposure, the DNA damage caused by both analogs was not significantly different at this time point. At later time points (8, 24, and 72 hr), d₄EDB caused significantly greater DNA damage than EDB. Since the decreased metabolism of d₄EDB was apparently due to a reduced rate of microsomal oxidation, these data support the hypothesis that conjugation with GSH is responsible for the genotoxic effects of EDB.     

Pb-inhibited mitotic activity in onion roots involves DNA damage and disruption of oxidative metabolism

Plant responses to abiotic stress significantly affect the development of cells, tissues and organs. However, no studies correlating Pb-induced mitotic inhibition and DNA damage and the alterations in redox homeostasis during root division per se were found in the literature. Therefore, an experiment was conducted to evaluate the impact of Pb on mitotic activity and the associated changes in the oxidative metabolism in onion roots. The cytotoxic effect of Pb on cell division was assessed in the root meristems of Allium cepa (onion). The mitotic index (MI) was calculated and chromosomal abnormalities were sought. Pb-treatment induced a dose-dependent decrease in MI in the onion root tips and caused mitotic abnormalities such as distorted metaphase, fragments, sticky chromosomes, laggards, vagrant chromosomes and bridges. Single Cell Gel Electrophoresis was also performed to evaluate Pb induced genotoxicity. It was accompanied by altered oxidative metabolism in the onion root tips suggesting the interference of Pb with the redox homeostasis during cell division. There was a higher accumulation of malondialdehyde, conjugated dienes and hydrogen peroxide, and a significant increase in the activities of superoxide dismutases, ascorbate peroxidases, guaiacol peroxidases and glutathione reductases in Pb-treated onion roots, whereas catalases activity exhibited a decreasing pattern upon Pb exposure. The study concludes that Pb-induced cytotoxicity and genotoxicity in the onion roots is mediated through ROS and is also tightly linked to the cell cycle. The exposure to higher concentrations arrested cell cycle leading to cell death, whereas different repair responses are generated at lower concentrations, thereby allowing the cell to complete the cell cycle.     

Inhibition of mitochondrial oxidative metabolism by SKF-525a in intact cells and isolated mitochondria

We have examined the effects of various concentrations of SKF-525A (β-diethylamino-ethyldiphenylpropyl acetate · HCl) on the energy metabolism of l and two types of ascites tumor cells, as well as on ion transport in liver slices. In liver slices, 0.2 to 1.0 mM SKF-525A caused an initial stimulation of O₂ uptake which was followed, at 0.5 to 1.0 mM, by a progressive inhibition of O₂ consumption, a fall of slice ATP content, and a reduced transport of K⁺, Na⁺ and Ca²⁺. In isolated mitochondria, we studied the effects of SKF525A on the rate of respiration and on the oxidation-reduction responses of NAD(P)⁺ and cytochrome b in the presence of various substrates. The results suggest that SKF-525A had three distinct actions on liver mitochondria, viz. an uncoupling action at low concentrations (0.02 to 0.17 mM); at higher concentrations (0.2 to 0.5 mM) an inhibition of the oxidation of NAD(P)⁺-linked substrates, exerted close to the substrate level; also at 0.2 to 0.5 mM, a less effective inhibition of electron transfer at a point between cytochrome b and O₂ in the electron-transfer chain. Experiments on O₂ consumption and cytochrome b oxidation-reduction changes in ascites cells showed only the first two of these effects in the intact tumor cells. We conclude that inhibition of mitochondrial energy-conserving reactions by SKF-525A can have a marked influence on energy-requiring aspects of liver-cell metabolism, one example of which is inhibition of cation active transport.     

Decrease of hepatic mitochondrial glutathione and mitochondrial injury induced by 1,2-dibromoethane in the rat in vivo: effect of diethylmaleate pretreatment

Diethylmaleate (DEM) potentiated the 1,2-dibromoethane (DBE)-induced hepatic morphological lesion in fasted male Wistar rats, as revealed by light and electron microscopy examination. The subcellular structures involved in such lesions were the mitochondria. The potentiating effect of DEM appeared to be due to enhancement of the depletion of hepatic mitochondrial glutathione (GSH) caused by DBE. DEM, however, failed to potentiate the DBE-induced release in the plasma of hepatic enzymes. The relationship between loss of mitochondrial GSH, mitochondrial injury, and the importance of the mitochondrial lesion in DBE-induced hepatotoxicity is discussed.     

Scopafungin, an inhibitor of oxidative phosphorylation in mitochondria

Scopafungin (U-29479), an antibiotic produced by a streptomycete strain, acts as an uncoupling agent of oxidative phosphorylation in mitochondria. In addition, mitochondrial respiration is also impaired but to a lesser degree. Studies of individual reaction sequences occurring within the respiratory chain and mitochondrial difference spectra suggest that scopafungin inhibits electron flow at the flavoprotein regions associated with the oxidation of NADH and succinate.     

The influence of disulfiram and other inhibitors of oxidative metabolism on the formation of 2-hydroxyethyl-mercapturic acid from 1,2-dibromoethane by the rat

The mercapturic acid derivative, $\text{N-}\text{acetyl}\text{-S-2-}\text{hydroxyethyl}\text{-}\text{l}\text{-}\text{cysteine}$, is a major metabolite of 1,2-dibromoethane in vivo. This compound can be formed via two pathways, both involving a potentially dangerous reactive intermediate. One way involves the intermediacy of bromoacetaldehyde, formed by microsomal oxidation, followed by loss of hydrogen bromide. The second pathway, direct conjugation of 1,2-dibromoethane with glutathione, gives rise to S-2-bromoethyl glutathione. Using several inhibitors of microsomal mixed function oxidases, it was found that under these conditions about 10% of the mercapturic acid derivative formed via direct conjugation.Disulfiram, an inhibitor of aldehyde dehydrogenases, but also of microsomal oxidation, also markedly inhibits the excretion of the mercapturic acid, after administration of a single high dose (1 g/kg) or upon chronic treatment with a low dose (50 mg/kg). The inhibitory effect is maximal after 10 days of chronic treatment. Administration of large amounts of 1,2-dibromoethane (>0.20 nmole/rat) following a single lower dose of disulfiram (125 mg/kg) also leads to a lower excretion of mercapturic acid metabolite a phenomenon associated with a decrease in cytochrome P-450 levels. From these results it is concluded that the enhanced carcinogenic effect of the combination disulfiram (chronic)/1,2-dibromoethane is not caused by bromoacetaldehyde, since its formation is completely inhibited under these conditions, but by S-2-bromoethyl-glutathione, although a role for 1,2-dibromoethane itself cannot be excluded.     

2-Ethylhexanol uncouples oxidative phosphorylation in rat liver mitochondria

2-Ethylhexanol (70 microM), a non-genotoxic carcinogen and peroxisome proliferator, stimulated oxygen uptake in the perfused rat liver by about 10% during the first 10 min of infusion. Perfusion with a higher, hepatotoxic dose of ethylhexanol (3 mM) led to a transient increase in oxygen uptake followed by a rapid inhibition of respiration of over 50% in 10 min. Lactate dehydrogenase (LDH) release, indicative of irreversible cell death, was detected in the effluent perfusate after 20 min. After 10 min of perfusion with ethylhexanol, livers were freeze-clamped, acid extracts were prepared and adenine nucleotides were measured by high-pressure liquid chromatography. Ethylhexanol decreased the ATP/ADP ratio from 2.5 to 0.9. Thus, marked decreases in hepatic energy state due to inhibition of respiration preceded cell death. To attempt to understand this phenomenon, the effect of ethylhexanol on isolated mitochondria was studied. Similar to classical uncoupling agents, ethylhexanol stimulated state-4 rates of respiration, diminished coupled rates of respiration, and decreased the P/O ratio in a dose-dependent manner in isolated mitochondria. Ethylhexanol also decreased uptake of radiolabeled 45CaCl2 by isolated mitochondria 4- to 5-fold. Therefore, we hypothesize that ethylhexanol initially uncouples oxidative phosphorylation leading to diminished ATP synthesis and collapse of ion gradients across the mitochondrial membrane.     

Carcinogenicity and toxicity of 1,2-dibromoethane in the rat

A chronic inhalation study of ethylene dibromide (EDB) was conducted in Sprague-Dawley rats. Four groups of rats received either control air, 0.05% disulfiram in the diet, 20 ppm of EDB, or 20 ppm of EDB and 0.05% disulfiram in the diet for 18 months. Rats receiving 20 ppm of EDB had high mortality and an increase, compared to controls, in one or both sexes of tumor incidences in the mammary gland, spleen, adrenal, liver, kidney, and subcutaneous tissue. A combined treatment of 20 ppm of EDB and 0.05% disulfiram in the diet resulted in earlier deaths (all within 14 months) and decreases in body weight gain, food consumption, hemoglobin, hematocrit, and RBC counts. This combination of EDB and disulfiram treatment also caused high incidences of tumors in liver, spleen, kidney, adrenal, thyroid, lung, mesentery, and mammary gland in one or both sexes. Testicular atrophy was found in 90% of the male rats that received a combination of EDB and disulfiram treatment.     

Extra-hepatic GSH-dependent metabolism of 1,2-dibromoethane (DBE) and 1,2-dibromo-3-chloropropane (DBCP) in the rat and mouse

Although DBE is metabolized by both microsomal and cytosolic pathways, it is the latter, GSH-dependent route, that may lead to hepatic and extra-hepatic genotoxicity and mutagenicity. As both DBE and DBCP exhibit predominantly extra-hepatic toxicity, their in vitro GSH-dependent debromination was measured in cytosolic fractions prepared from liver, kidney, testes and stomachs of Sprague-Dawley rats and Swiss-Webster mice. There was a marked species difference between the rat and mouse, with the rat metabolizing DBCP more rapidly than DBE, and the mouse metabolizing DBE more rapidly than DBCP. Hepatic rates exceeded those seen in extra-hepatic tissues in every case. Extra-hepatic rates of debromination represented as much as 84% of the hepatic rates, and generally followed the order: kidney greater than testes greater than stomach. Rates of metabolism for DBE and DBCP represented only a small fraction of the total cytosolic GSH S-transferase activity. These findings suggest significant levels of GSH-dependent metabolism may occur within those tissues associated with the in vivo toxicity of DBE and DBCP.     

Effects of sesquiterpene lactones on mitochondrial oxidative phosphorylation

1. The toxic plant sesquiterpene lactones, helenalin, hymenoxon, mexicanin-E, tenulin, dihydrogriesenin, and psilotropin which were isolated from Helenium, Hymenoxys, and Geigeria spp markedly inhibited "state 3" respiration in mouse hepatic mitochondria. With the exception of dihydrogriesenin and psilotropin, all the other sesquiterpene lactones stimulated "state 4" respiration. 2. The sesquiterpene lactones also stimulated ATPase activity in the presence of Mg2+ ions and caused mitochondrial swelling in buffer solutions containing magnesium, sodium, ammonium and potassium chloride salts. 3. The number of alkylating sites present in a sesquiterpene lactone appears to be related to the inhibitory activity on mitochondrial oxidative phosphorylation, however, the structure-activity relationship of these lactones is not clear at present. 4. Mitochondria prepared from the liver of ethoxyquin hydrochloride-fed mice were less susceptible to sesquiterpene lactone-mediated inhibition of mitochondrial oxidative phosphorylation.     

Di-n-butyltindichloride uncouples oxidative phosphorylation in rat liver mitochondria

In this study di-n-butyltindichloride (DBTC) was found to inhibit α-ketoacid-stimulated response of rat liver mitochondria to the addition of ADP and the uncoupler carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). The α-ketoglutarate oxidation was already inhibited at a level of 0.8 nmol DBTC/mg protein. When succinate was used as substrate together with rotenone, the State 3 and FCCP stimulated oxidation were not inhibited by DBTC. But from a level of 8.3 nmol DBTC/mg protein, the State 4 respiration was increased. It is concluded that in low amounts DBTC specifically blocks α-ketoacid dehydrogenases, but higher concentrations of this compound uncouples oxidative phosphorylation. However, this uncoupling will be masked when the NADH production from substrate oxidation is decreased by DBTC as will be in case of α-ketoacids.     

The mitochondrial metabolism of 1,2-disubstituted hydrazines, procarbazine and 1,2-dimethylhydrazine

Sonication of isolated rat hepatocytes caused a pronounced decrease in the metabolism of biphenyl due to dilution of the cytosolic pool of NADPH, but did not greatly reduce the rate of procarbazine oxidation to its azo derivative. This result suggested the existence of two enzyme systems which can oxidize 1,2-disubstituted hydrazines: an NADPH-dependent (cytochrome P-450) and an NADPH-independent hydrazine oxidase. Upon assaying the various cell fractions of the hepatocyte, it was noted that the NADPH-independent hydrazine oxidase activity was localized in the mitochondria. The reaction was not linked to mitochondrial electron transport, but preincubation of isolated mitochondria with N,N-dimethylpropargylamine markedly inhibited both monoamine oxidase activity (benzylamine and kynuramine deamination) and procarbazine oxidation. During a 15-fold purification of the enzyme, benzylamine oxidase and procarbazine oxidase activity copurified, demonstrating that the rat liver mitochondrial monoamine oxidase can convert procarbazine to its respective azo derivative. 1,2-Dimethyl- and monomethylhydrazine were also metabolized by monoamine oxidase. Procarbazine did not inactivate monoamine oxidase like other hydrazines; this most likely reflects the fact that the oxidation product of the 1,2-disubstituted hydrazines is a stable azo derivative. However, procarbazine is a competitive substrate for the enzyme and may exert some of its toxic neurological effects by altering the metabolism of biogenic amines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct quantification of mitochondria and mitochondrial DNA dynamics

Mitochondria are known to be one of major organelles within a cell and to play a crucial role in many cellular functions. These organelles show the dynamic behaviors such as fusion, fission and the movement along cytoskeletal tracks. Besides mitochondria, mitochondrial DNA is also highly motile. Molecular analysis revealed that several proteins are involved in mitochondria and mitochondrial DNA dynamics. In addition to the degeneration of specific nerves with high energy requirement, mutation of genes coding these proteins results in metabolic diseases. During the last few years, a significant amount of relevant data has been obtained on molecular basis of these diseases but mitochondrial dynamics in cells derived from the patients is poorly understood. So far time-lapse fluorescence microscopy, fluorescence recovery after photo bleaching and image correlation methods have been used to study organellar motion. Especially, image correlation method has possibility to evaluate diffusion coefficient of mitochondria and mitochondrial DNA simultaneously and directly. When we search candidates for compounds that modulate mitochondrial dynamics by high throughput screening, image correlation method may be useful although the careful interpretation is required for crowded and heterogeneous environment within a cell.     

Uncoupling of oxidative phosphorylation in rat liver mitochondria by chloroethanols.

Chloroethanols are toxic chemicals used in industry and also formed as a result of the metabolism of several widely used halogenated hydrocarbons. The effect of 2-chloroethanol (CE), 2,2-dichloroethanol (DCE) and 2,2,2-trichloroethanol (TCE) on rat liver mitochondrial respiration was studied. Rat liver mitochondria were isolated in a medium consisting of 250 mM sucrose, 10mM Tris-HCl and 1 mM EDTA (pH 7.4). Mitochondrial respiration was determined with an oxygen electrode at 30 degrees C and the polarographic buffer consisted of 250 mM mannitol, 10 mM KCl, 10 mM K2HPO4, 5 mM MgCl2, 0.2 mM EDTA and 10 mM Tris-HCl (pH 7.4). With succinate as the respiratory substrate and using chloroethanols (150 mM), CE stimulated respiration by 28.2 +/- 6.5% and DCE by 202.7 +/- 8.2% while TCE inhibited mitochondrial respiration (greater than 95%). The effect of change in the concentration of chloroethanols on mitochondrial respiration was also studied. CE showed maximum stimulation at 600 mM (97.6%), DCE at 150 mM (202.6%) and TCE at 30 mM (313.6%). Respiratory stimulation was independent of mitochondrial protein concentration. Chloroethanols (optimal concentrations for respiratory stimulation with succinate) inhibited mitochondrial respiration when glutamate-malate was used as the respiratory substrate. Estimation of adenosine triphosphate (ATP) showed that chloroethanols inhibited the synthesis of ATP. These results indicate that chloroethanols stimulate mitochondrial respiration by uncoupling oxidative phosphorylation and that the uncoupling potency is proportional to the extent of chlorination at the beta-position of haloethanol.     

Tacrolimus and sirolimus decrease oxidative phosphorylation of isolated rat kidney mitochondria

1. Tacrolimus and sirolimus are potent immunosuppressors used in transplantation. Tacrolimus has been suspected to alter mitochondrial respiration of different tissues but sirolimus has not been evaluated. 2. We evaluated the in vitro effect of tacrolimus and sirolimus on oxidative phosphorylation of isolated rat kidney mitochondria. 3. Oxygen consumption was measured with a Clark-type electrode. Tacrolimus and sirolimus increased the resting rate (state 4) and had no significant effect on ADP-stimulated respiration (state 3). The decrease of respiratory control ratio was concentration-dependent with a biphasic curve for tacrolimus. The EC(50)s were 3.4 x 10(-11) M and 2.3 x 10(-8) M for tacrolimus and 4.4 x 10(-10) M for sirolimus. The maximal inhibition was 20 and 14% for tacrolimus and sirolimus, respectively. 4. Tacrolimus and sirolimus had an uncoupling effect on oxidative phosphorylation related to a decrease of the inner membrane fluidity. At the opposite of cyclosporin A, no effect on swelling or Ca(2+) fluxes was observed. 5. All events occurred at therapeutic concentrations and then could appear during long-term treatment. Cellular consequences such as chronic nephrotoxicity with tacrolimus are suggested. The risk of cyclosporin A nephrotoxicity potentiation by sirolimus is discussed.     

Inhibition of oxidative phosphorylation in tumor cells and mitochondria by daunomycin and adriamycin

The binding of copper to daunomycin has been investigated. It is concluded that the strenght of binding is not large enough for the 1:2 copper-daunomycin complex to exist in vivo. Daunomycin and adriamycin inhibit glutamate- and pyruvate-malate-dependent oxidative phosphorylation in bovine heart mitochondria and adriamycin uncouples this process as well. No inhibition of Ehrlich ascites tumor cell or mitochondrial respiration by daunomycin is observed at concentrations much larger than those used for heart. In conjunction with the work of others, these results suggest a role for the inhibition of oxidative phosphorylation in the cardiac toxicity of these anthracycline drugs.     

Interaction of butylated hydroxyanisole with mitochondrial oxidative phosphorylation.

The antioxidant, butylated hydroxyanisole (BHA), has a number of effects on mitochondrial oxidative phosphorylation. In this study we apply the novel approach developed by Brand (Brand MD, Biochim Biophys Acta 1018: 128-133, 1990) to investigate the site of action of BHA on oxidative phosphorylation in rat liver mitochondria. Using this approach we show that BHA increases the proton leak through the mitochondrial inner membrane and that it also inhibits the delta p (proton motive force across the mitochondrial inner membrane) generating system, but has no effect on the phosphorylation system. This demonstrates that compounds having pleiotypic effects on mitochondrial oxidative phosphorylation in vitro can be analysed and their many effects distinguished. This approach is of general use in analysing many other compounds of pharmacological interest which interact with mitochondria. The implications of these results for the mechanism of interaction of BHA with mitochondrial oxidative phosphorylation are discussed.     

Indirubin-3'-oxime impairs mitochondrial oxidative phosphorylation and prevents mitochondrial permeability transition induction

Indirubin, a red colored 3,2′-bisindole isomer, is a component of Indigo naturalis and is an active ingredient used in traditional Chinese medicine for the treatment of chronic diseases. The family of indirubin derivatives, such as indirubin-3′-oxime, has been suggested for various therapeutic indications. However, potential toxic interactions such as indirubin effects on mitochondrial bioenergetics are still unknown. This study evaluated the action of indirubin-3′-oxime on the function of isolated rat liver mitochondria contributing to a better understanding of the biochemical mechanisms underlying the multiple effects of indirubin. Indirubin-3′-oxime incubated with isolated rat liver mitochondria, at concentrations above 10μM, significantly depresses the phosphorylation efficiency of mitochondria as inferred from the decrease in the respiratory control and ADP/O ratios, the perturbations in mitochondrial membrane potential and in the phosphorylative cycle induced by ADP. Furthermore, indirubin-3′-oxime at up to 25μM stimulates the rate of state 4 respiration and inhibits state 3 respiration. The increased lag phase of repolarization was associated with a direct inhibition of the mitochondrial ATPase. Indirubin-3′-oxime significantly inhibited the activity of complex II and IV thus explaining the decreased FCCP-stimulated mitochondrial respiration. Mitochondria pre-incubated with indirubin-3′-oxime exhibits decreased susceptibility to calcium-induced mitochondrial permeability transition. This work shows for the first time multiple effects of indirubin-3′-oxime on mitochondrial bioenergetics thus indicating a potential mechanism for indirubin-3′-oxime effects on cell function.