Oxidative metabolism in rat hepatocytes and mitochondria during sepsis

We hypothesized that cellular oxygen consumption is abnormal during sepsis as a result of increased oxidative stress and selective mitochondrial damage. In a rat model of sepsis (cecal ligation and puncture), we studied the respiratory characteristics of isolated hepatocytes and liver mitochondria 16 h after onset of septic injury. Endogenous respiration by isolated cells was decreased during sepsis, while cyanide-resistant (nonmitochondrial) respiration was unaffected. Maximal oxygen consumption in ADP-supplemented, permeabilized hepatocytes was decreased with succinate as the substrate, but not with malate + glutamate or TMPD + ascorbate. In contrast, maximum oxygen consumption (State 3) by isolated liver mitochondria increased up to 35% during sepsis using either succinate or malate + glutamate as substrate. The electrophoretic features and mobility of nondenatured mitochondrial respiratory complexes were similar in control and septic hepatocytes, with the exception of decreased Complex V protein in sepsis. Structural evaluation of mitochondria in fixed liver slices by electron microscopy showed mitochondrial swelling in most of the septic animals. Measurements of oxidative stress during sepsis suggested an increase in hydroxylation of salicylate by isolated hepatocytes, and mitochondrial protein carbonyl content was increased significantly. Induction of iNOS in hepatocytes after 16 h of sepsis was variable, and little release of the oxidation products of NO. was detected. These findings are interpreted to mean that hepatocytes contain a mixed population of injured and hyperfunctional mitochondria during sepsis.     

The effect of idebenone, a coenzyme Q analogue, on hydrophobic bile acid toxicity to isolated rat hepatocytes and hepatic mitochondria

Oxidant stress induced by hydrophobic bile acids has been implicated in the pathogenesis of liver injury in cholestatic liver disorders. We evaluated the effect of idebenone, a coenzyme Q analogue, on taurochenodeoxycholic acid (TCDC)-induced cell injury and oxidant stress in isolated rat hepatocytes and on glycochenodeoxycholic acid (GCDC)-induced generation of hydroperoxides in fresh hepatic mitochondria. Isolated rat hepatocytes in suspension under 9% oxygen atmosphere were preincubated with 0, 50, and 100 micromol/l idebenone for 30 min and then exposed to 1000 micromol/l TCDC for 4 h. LDH release (cell injury) and thiobarbituric acid reactive substances (measure of lipid peroxidation) increased after TCDC exposure but were markedly suppressed by idebenone pretreatment. In a second set of experiments, the addition of 100 micromol/l idebenone up to 3 h after hepatocytes were exposed to 1000 micromol/l TCDC resulted in abrogation of subsequent cell injury and markedly reduced oxidant damage to hepatocytes. Chenodeoxycholic acid concentrations increased to 5.15 nmol/10(6) cells after 2 h and to 7.05 after 4 h of incubation of hepatocytes with 1000 micromol/l TCDC, and did not differ in the presence of idebenone. In freshly isolated rat hepatic mitochondria, when respiration was stimulated by succinate, 10 micromol/l idebenone abrogated the generation of hydroperoxides during a 90-minute exposure to 400 micromol/l GCDC. These data demonstrate that idebenone functions as a potent protective hepatocyte antioxidant during hydrophobic bile acid toxicity, perhaps by reducing generation of oxygen free radicals in mitochondria.     

The dependence on the extramitochondrial ATP/ADP-ratio of the oxidative phosphorylation in mitochondria isolated by a new procedure from rat skeletal muscle

A simple method for preparation of rat skeletal muscle mitochondria is presented using gentle mechanical homogenization in a syringe and nagarse treatment (EC 3.4.4.16). This method enables the preparation of skeletal muscle mitochondria, whose outer membrane is intact to 95%. Furthermore, with mitochondria prepared by this method the regulation of respiration and phosphorylation by the extramitochondrial ATP/ADP-ratio can be demonstrated. In accordance to rat liver and heart mitochondria and to mitochondria of rabbit reticulocytes, the regulation by the extramitochondrial ATP/ADP-ratio lies in the range from 5 (corresponding to 98% of the maximum respiration) to 100 (corresponding to state 4). At extramitochondrial ATP/ADP-ratios from 0.01 to 1 the respiration rate is nearly constant (maximum rate of respiration).     

Mitochondrial effects of L-ropivacaine, a new local anesthetic

The effects of the local anesthetics ropivacaine and bupivacaine were investigated on isolated rat liver mitochondria. The efficiency of oxidative phosphorylation was evaluated by measuring the rates of respiration and ATP synthesis and the magnitude of the transmembrane electrical potential (deltapsi). Bupivacaine did not alter the ADP-stimulated respiration but strongly affected the resting respiration, which was more than doubled at 0.6 mM. In addition, it decreased the transmembrane electrical potential, and the ATP synthesis rate (deltapsi was less than 100 mV at 0.6 mM). Ropivacaine did not alter the ADP-stimulated respiration, and the resting respiration seemed to be substantially unaffected up to 1.2 mM; a slight increase was observed at 1.8 and 2.4 mM. The transmembrane potential was decreased by anesthetic concentrations higher than 1.2 mM and ATP synthesis was consequently affected. The findings suggest that ropivacaine is less toxic than bupivacaine, in rat liver mitochondria.     

Direct effects of diazoxide on mitochondria in pancreatic B-cells and on isolated liver mitochondria

1. The direct effects of diazoxide on mitochondrial membrane potential, Ca2+ transport, oxygen consumption and ATP generation were investigated in mouse pancreatic B-cells and rat liver mitochondria. 2. Diazoxide, at concentrations commonly used to open adenosine 5'-triphosphate (ATP)-dependent K+-channels (K(ATP) channels) in pancreatic B-cells (100 to 1000 microM), decreased mitochondrial membrane potential in mouse intact perifused B-cells, as evidenced by an increase of rhodamine 123 fluorescence. This reversible decrease of membrane potential occurred at non-stimulating (5 mM) and stimulating (20 mM) glucose concentrations. 3. A decrease of mitochondrial membrane potential in perifused B-cells was also caused by pinacidil, but no effect could be seen with levcromakalim (500 microM each). 4. Measurements by a tetraphenylphosphonium-sensitive electrode of the membrane potential of rat isolated liver mitochondria confirmed that diazoxide decreased mitochondrial membrane potential by a direct action. Pretreatment with glibenclamide (2 microM) did not antagonize the effects of diazoxide. 5. In Fura 2-loaded B-cells perifused with the Ca2+ channel blocker, D 600, a moderate, reversible increase of intracellular Ca2+ concentration could be seen in response to 500 microM diazoxide. This intracellular Ca2+ mobilization may be due to mitochondrial Ca2+ release, since the reduction of membrane potential of isolated liver mitochondria by diazoxide was accompanied by an accelerated release of Ca2+ stored in the mitochondria. 6. In the presence of 500 microM diazoxide, ATP content of pancreatic islets incubated in 20 mM glucose for 30 min was significantly decreased by 29%. However, insulin secretion from mouse perifused islets induced by 40 mM K+ in the presence of 10 mM glucose was not inhibited by 500 microM diazoxide, suggesting that the energy-dependent processes of insulin secretion distal to Ca2+ influx were not affected by diazoxide at this concentration. 7. The effects of diazoxide on oxygen consumption and ATP production of liver mitochondria varied depending on the respiratory substrates (5 mM succinate, 10 mM alpha-ketoisocaproic acid, 2 mM tetramethyl phenylenediamine plus 5 mM ascorbic acid), indicating an inhibition of respiratory chain complex II. Pinacidil, but not levcromakalim, inhibited alpha-ketoisocaproic acid-fuelled ATP production. 8. In conclusion, diazoxide directly affects mitochondrial energy metabolism, which may be of relevance for stimulus-secretion coupling in pancreatic B-cells.     

The utilisation of creatine and its analogues by cytosolic and mitochondrial creatine kinase

We have investigated the utilisation of four analogues of creatine by cytosolic Creatine Kinase (CK), using 31P-NMR in the porcine carotid artery, and by mitochondrial CK (Mt-CK), using oxygen consumption studies in isolated heart mitochondria and skinned fibers. Porcine carotid arteries were superfused for 12 h with Krebs-Henseleit buffer at 22 degrees C, containing 11 mM glucose as substrate, and supplemented with either 20 mM beta-guanidinopropionic acid (beta-GPA), methyl-guanidinopropionic acid (m-GPA), guanidinoacetic acid (GA) or cyclocreatine (cCr). All four analogues entered the tissue and became phosphorylated by CK as seen by 31 P-NMR, Inhibition of oxidative metabolism by 1 mM cyanide after accumulation of the phosphorylated analogue resulted in the utilisation of PCr, beta-GPA-P, GA-P and GA-P over a similar time course (approximately 2 h), despite very different kinetic properties of these analogues in vitro. cCr-P was utilised at a significantly slower rate, but was rapidly dephosphorylated in the presence of both 1 mM iodoacetate and cyanide (to inhibit both glycolysis and oxidative metabolism respectively). The technique of creatine stimulated respiration was used to investigate the phosphorylation of the analogues by Mt-CK, Isolated mitochondria were subjected to increasing [ATP], whereas skinned fibres received a similar protocol with increasing [ADP]. There was a significant stimulation of respiration by creatine and cCr in isolated mitochondria (decreased K(m) and increased Vmax vs control), but none by GA, mGPA or beta-GPA (also in skinned fibres), indicating that these latter analogues were not utilised by Mt-CK. These results demonstrate differences in the phosphorylation and dephosphorylation of creatine and its analogues by cytosolic CK and Mt-CK in vivo and in vitro.     

The involvement of cytochrome P450 peroxidase in the metabolic bioactivation of cumene hydroperoxide by isolated rat hepatocytes

Organic hydroperoxides are believed to be primarily detoxified in cells by the GSH peroxidase/GSSG reductase system and activated to cytotoxic radical species by non-heme iron. However, organic hydroperoxides seem to be bioactivated by cytochrome P450 (P450) in isolated hepatocytes as various P450 (particularly P450 2E1) inhibitors inhibited cumene hydroperoxide (CumOOH) metabolism and attenuated subsequent cytotoxic effects including antimycin A-resistant respiration, lipid peroxidation, iron mobilization, ATP depletion, and cell membrane disruption. CumOOH metabolism was also faster in P450 1A-induced hepatocytes and was inhibited by the P450 1A inhibitor alpha-naphthoflavone. The ferric chelator deferoxamine also prevented cytotoxicity even after CumOOH had been metabolized but had no effect on CumOOH metabolism. This emphasizes the toxicological significance of the iron released following hydroperoxide metabolic activation by cytochrome P450. The radical trap, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), had no effect on CumOOH metabolism but prevented CumOOH-induced antimycin A-resistant respiration, lipid peroxidation, iron mobilization, and loss of membrane integrity. These results suggest that CumOOH is metabolically activated by some P450 enzymes (e.g., P450 2E1) in hepatocytes to form reactive radical metabolites or oxidants that cause lipid peroxidation and cytotoxicity.     

In vivo heat shock protects rat myocardial mitochondria

Heat shock (HS)/stress proteins (HSP) provide protection from a variety of stresses other than HS, including oxidative stress and mitochondria have been implicated as the target of HS-related protection in stressed cultured cells. Here we investigated whether mitochondria also are targets for the HS-mediated protection in vivo. Sprague Dawley rats were exposed, or not, to HS (41 degrees C, 15 min). After a 21 h recovery period, hearts were excised and perfused with or without H2O2 (0.15 mM). Myocardial mitochondria were then isolated, and their oxygen consumption was analyzed. HS prevented H2O2-induced alterations in state 3 respiration while increasing the expression of Hsp70 and heme oxygenase (HO). Thus, in vivo HS protects rat myocardial mitochondrial respiration against the deleterious effects of oxidative injury, a protection relating to Hsp70 and/or HO and targeting state 3 respiration.     

In vitro and in vivo protective effect of honokiol on rat liver from peroxidative injury

Honokiol, a compound extracted from the Chinese medicinal herb Magnolia officinalis, has a strong antioxidant effect on the inhibition of lipid peroxidation in rat heart mitochondria. To investigate the protective effect of honokiol on hepatocytes from peroxidative injury, oxygen consumption and malondialdehyde formation for in vitro iron-induced lipid peroxidation were assayed, and the mitochondrial respiratory function for in vivo ischemia-reperfusion injury were evaluated in rat liver, respectively. The inhibitory effect of honokiol on oxygen consumption and malondialdehyde formation during iron-induced lipid peroxidation in liver mitochondria showed obvious dose-dependent responses with a concentration of 50% inhibition being 2.3 x 10(-7) M and 4.96 x 10(-7) M, respectively, that is, 550 times and 680 times more potent than alpha-tocopherol, respectively. When rat livers were introduced with ischemia 60 min followed by reperfusion for 60 min, and then pretreated with honokiol (10 micrograms/kg BW), the mitochondrial respiratory control ratio (the quotient of the respiration rate of State 3 to that of State 4) and ADP/O ratio from the honokiol-treated livers were significantly higher than those of non-treated livers during reperfusion. The dose-dependent protective effect of honokiol on ischemia-reperfusion injury was 10 microgram-100 micrograms/Kg body weight. We conclude that honokiol is a strong antioxidant and shed insight into clinical implications for protection of hepatocytes from ischemia-reperfusion injury.     

Starting at the beginning, middle, and end: translation initiation in eukaryotes

A method is described for the densitometric determination of the p-hydroxybenzoic esters and p-hydroxybenzoic acid in mixtures or in drugs. This method is compared with the one used in high performance liquid chromatography (HPLC). The calibration curves were linear in interval 0.250-3.60 mumol ml-1 per 200 nl per spot. The limit of detection and the relative standard deviation (RSD) are higher than in HPLC (RSD is 6% in HPTLC. 3% in HPLC; limit of detection about 40 pmol in HPTLC and 25 pmol in HPLC) but HPTLC quantitative determination of parabens in drugs is faster.     

Linoleic acid-induced activity of plant uncoupling mitochondrial protein in purified tomato fruit mitochondria during resting, phosphorylating, and progressively uncoupled respiration

An uncoupling protein was recently discovered in plant mitochondria and demonstrated to function similarly to the uncoupling protein of brown adipose tissue. In this work, green tomato fruit mitochondria were purified on a self-generating Percoll gradient in the presence of 0.5% bovine serum albumin to deplete mitochondria of endogenous free fatty acids. The uncoupling protein activity was induced by the addition of linoleic acid during the resting state, and in the progressively uncoupled state, as well as during phosphorylating respiration in the presence of benzohydroxamic acid, an inhibitor of the alternative oxidase and with succinate (+ rotenone) as oxidizable substrate. Linoleic acid strongly stimulated the resting respiration in fatty acid-depleted mitochondria but had no effect on phosphorylating respiration, suggesting no activity of the uncoupling protein in this respiratory state. Progressive uncoupling of state 4 respiration decreased the stimulation by linoleic acid. The similar respiratory rates in phosphorylating and fully uncoupled respiration in the presence and absence of linoleic acid suggested that a rate-limiting step on the dehydrogenase side of the respiratory chain was responsible for the insensitivity of phosphorylating respiration to linoleic acid. Indeed, the ADP/O ratio determined by ADP/O pulse method was decreased by linoleic acid, indicating that uncoupling protein was active during phosphorylating respiration and was able to divert energy from oxidative phosphorylation. Moreover, the respiration rates appeared to be determined by membrane potential independently of the presence of linoleic acid, indicating that linoleic acid-induced stimulation of respiration is due to a pure protonophoric activity without any direct effect on the electron transport chain.     

Plasmatocyte spreading peptide is encoded by an mRNA differentially expressed in tissues of the moth Pseudoplusia includens

We investigated in rats the effect of 4 wk of hypodynamia on the respiration of mitochondria isolated from four distinct muscles [soleus, extensor digitorum longus, tibial anterior, and gastrocnemius (Gas)] and from subsarcolemmal (SS) and intermyofibrillar (IMF) regions of mixed hindlimb muscles that mainly contained the four cited muscles. With pyruvate plus malate as respiratory substrate, 4 wk of hindlimb suspension produced an 18% decrease in state 3 respiration for IMF mitochondria compared with those in the control group (P < 0.05). The SS mitochondria state 3 were not significantly changed. Concerning the four single muscles, the mitochondrial respiration was significantly decreased in the Gas muscle, which showed a 59% decrease in state 3 with pyruvate + malate (P < 0.05). The other muscles presented no significant decrease in respiratory rate in comparison with the control group. With succinate + rotenone, there was no significant difference in the respiratory rate compared with the respective control group, whatever the mitochondrial origin (SS, or IMF, or from single muscle). We conclude that 4 wk of hindlimb suspension alters the respiration of IMF mitochondria in hindlimb skeletal muscles and seems to act negatively on complex I of the electron-transport chain or prior sites. The muscle mitochondria most affected are those isolated from the Gas muscle.     

Modulation of oxidative phosphorylation by Mg2+ in rat heart mitochondria

The effect of varying the Mg2+ concentration on the 2-oxoglutarate dehydrogenase (2-OGDH) activity and the rate of oxidative phosphorylation of rat heart mitochondria was studied. The ionophore A23187 was used to modify the mitochondrial free Mg2+ concentration. Half-maximal stimulation (K0.5) of ATP synthesis by Mg2+ was obtained with 0.13 +/- 0.02 mM (n = 7) with succinate (+rotenone) and 0.48 +/- 0.13 mM (n = 6) with 2-oxoglutarate (2-OG) as substrates. Similar K0.5 values were found for NAD(P)H formation, generation of membrane potential, and state 4 respiration with 2-OG. In the presence of ADP, an increase in Pi concentration promoted a decrease in the K0.5 values of ATP synthesis, membrane potential formation and state 4 respiration for Mg2+ with 2-OG, but not with succinate. These results indicate that 2-OGDH is the main step of oxidative phosphorylation modulated by Mg2+ when 2-OG is the oxidizable substrate; with succinate, the ATP synthase is the Mg2+-sensitive step. Replacement of Pi by acetate, which promotes changes on intramitochondrial pH abolished Mg2+ activation of 2-OGDH. Thus, the modulation of the 2-OGDH activity by Mg2+ has an essential requirement for Pi (and ADP) in intact mitochondria which is not associated to variations in matrix pH.     

cAMP and Ca2+ involvement in the mitochondrial response of cultured fetal rat hepatocytes to adrenaline

The effect of adrenaline on the control of respiratory activity of mitochondria from fetal hepatocytes in primary culture was studied. In the absence of adrenaline, the respiratory control ratio (RCR) of mitochondria increased during the first 3 days of culture due to a decrease in the rate of state 4 respiration. The presence of adrenaline in the incubation medium further increased the mitochondrial RCR through a decrease in the rate of respiration in state 4 and to an increase in the respiration rate in state 3. The effect of adrenaline was mimicked by dibutyryl-cAMP, forskolin, and isobutyl methyl xanthine. All these compounds increased cAMP concentrations, suggesting that cAMP may be involved in the effect of adrenaline. The increase in intracellular free Ca2+ concentrations caused by phenylephrine, vasopressin, or thapsigargin was also accompanied by an increase in the RCR, suggesting that both phenomena are associated. Dibutyryl-cAMP also increased free Ca2+ concentrations, suggesting that the effects of cAMP may be mediated by free Ca2+ concentrations. Adrenaline, dibutyryl-cAMP, phenylephrine, vasopressin, and thapsigargin promoted adenine nucleotide accumulation in mitochondria; this may be an intermediate step in the activation of mitochondrial respiratory function. These results suggest that the stimulatory effect of adrenaline on mitochondrial maturation in cultured fetal rat hepatocytes may be exerted through a mechanism in which both cAMP and Ca2+ act as second messengers. It is concluded that the effect of adrenaline on mitochondrial maturation is exerted by both alpha- and beta-adrenergic mechanisms and is mediated by the increase in adenine nucleotide contents of mitochondria.     

An assessment of fertility in boron-exposed Turkish subpopulations

Treatment after hypoxia-ischemia (HI) in immature rats with the N-methyl-D-aspartate receptor (NMDAR) antagonist dizocilpine maleate (MK-801) reduces areas with high glucose utilization and reduces brain damage. The object was to study the metabolic effects of MK-801 treatment after HI. Seven-day-old rats were randomized to the following groups: non-HI, HI, or HI plus MK-801 (0.5 mg/kg immediately after HI). In the parietal cortex, the mitochondrial respiration was measured in homogenates 1 to 4 hours, and the energy metabolites at 3 and 8 hours after HI. The energy use was calculated from changes in energy metabolites after decapitation at 3 hours after HI. State 3 respiration was reduced by 46%, 32%, and 25% after HI compared with non-HI with pyruvate plus malate, glutamate plus malate, or glutamate plus succinate as substrates, respectively. Uncoupler-stimulated but not state 4 respiration was similarly reduced. The MK-801 augmented pyruvate plus malate-supported state 3 respiration after HI by 42%. The energy utilization was not affected by HI but was reduced by MK-801 treatment in the ipsilateral cortex from 4.6 +/- 2.3 to 2.6 +/- 1.8 micromol high-energy phosphate bond/min/g. The levels of ATP and phosphocreatine did not differ between the HI and HI plus MK-801 groups at 3 hours, but were lower in the HI than in the HI plus MK-801 group at 8 hours after HI. In conclusion, treatment with MK-801 reduced energy utilization and improved mitochondrial function and energy status after HI, suggesting a linkage between NMDAR activation and impaired energy metabolism during reperfusion.     

The use of cultured hepatocytes to investigate the mechanisms of drug hepatotoxicity

In the course of biotransformation reactions catalyzed both by cytochrome P450 and by conjugating enzymes, drug-derived reactive metabolites and active oxygen species can appear that may escape the detoxification process, initiating radical chain reactions (e.g., lipid peroxidation), covalently binding to macromolecules (proteins, DNA), or impairing the energetic balance of cells. This is usually followed by alterations of ion homeostasis that precede irreversible biochemical changes and cell death. There are, however, cellular mechanisms of defense that prevent, or repair, the damage caused by these reactive intermediates. Ultimately it is the balance between bioactivation, detoxification, and defense mechanisms that determines whether a compound will or will not elicit a toxic effect. Cultures of hepatocytes, including those of human origin, can be used to elucidate the mechanisms of drug toxicity. This is illustrated in the study of the mechanism of hepatotoxicity by diclofenac. Much less cytotoxicity is observed in nonmetabolizing hepatomas than in hepatocytes. The observed cell dysfunction parallels the biotransformation of the drug, and particularly the formation of the minor metabolite N,5-dihydroxydiclofenac by hepatocytes. This compound is able to inhibit mitochondrial ATP synthesis in hepatocytes.     

Respiration and ion permeability of the inner membrane in rat "sodium" liver mitochondria

Ion permeability of internal membrane and a respiration in isolated rat liver mitochondria, further related to as "sodium ones", were studied following replacement of K+ ions for Na+ ones in the mitochondrial matrix. As compared with the control ("potassium mitochondria"), state 4 respiration in the sodium mitochondria, energized by succinate, was shown to be enhanced in KCl or sucrose media. Oxygen consumption rates in the sodium mitochondria, being in state 3 or stimulated by 2,4-dinitrophenol, were lower than rates for the control mitochondria. This effect was much pronounced in the sucrose medium. The coefficients, characterizing the distribution of 137Cs between mitochondria and the medium, were lower for the sodium mitochondria than for the control in the presence of 2.5 mM succinate and 10(-8) M valinomycin. In comparison with the control, a more extensive swelling for the sodium mitochondria was found, first, in the medium containing 25 mM K-acetate and 100 mM sucrose for succinate-energized mitochondria, and second, in the medium containing 125 mM NH4NO3 without mitochondrial energization. Changes disclosed in respiration, swelling and coefficients of 137Cs distribution for the sodium mitochondria are supposed to be caused by non-uniform effects of Na+ and K+ ions on the water structure of mitochondrial matrix, ion permeability of internal membrane, and the activity in oxidative phosphorylation enzymes.     

Urban-rural difference in cereal consumption by people in Shandong Province, China

The influence of the 1,4-dihydropyridines (DHPs), water-soluble glutapyrone available as sodium, potassium and ammonium salts of 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-DHP-4-carboxamide)glutaric acid, from one side, and a lipophylic cerebrocrast, 2-propoxyethyl 2,6-dimethyl-4-(2-difluoromethoxyphenyl)-1,4-DHP-3,5-dicarboxylate, from the other side, on partially damaged mitochondria of the Wistar rat hindlimb muscle was also studied. The following tests were made: (1) rates of endogenous respiration and substrate (succinate) oxidation and oxidative phosphorylation; (2) rates and amplitudes of high-amplitude swelling and contraction after the addition of ATP, ADP and succinate to the previously swollen mitochondria and (3) rate of reversible self-aggregation of mitochondria isolated in salt media after ATP-induced contraction without and in the presence of azidothymidine (AZT). Cerebrocrast (10-100 microM) partially normalized the endogenous respiration rate and slightly augmented the respiration rate after the addition of succinate and to lesser extent ADP. Cerebrocrast in a concentration-dependent manner (2.5-50 microM) increased (two-fold at 20-50 microM) the active contraction amplitude of swollen mitochondria, induced by single or repeated additions of ATP. The influence of cerebrocrast on the ADP- and succinate-induced contractions was less obvious. Unlike cerebrocrast glutapyrone caused a reduction of the ATP-induced contraction amplitude (two-fold at 0.5-5.0 mM), not impairing the mitochondrial contraction ability in response to ATP or succinate. Pre-exposure to 2.5 mM glutapyrone resulted in at least a 10-fold inhibition of the reversible aggregation rate in the presence of 99 and 198 microM AZT. The results suggest the usefulness of further study of cerebrocrast and glutapyrone in preventing AZT-induced and some other mitochondrial myopathies.     

Nephrotoxicity and hepatotoxicity of 5,6-dichloro-4-thia-5-hexenoic acid: evidence for fatty acid beta-oxidation-dependent bioactivation

5,6-Dichloro-4-thia-5-hexenoic acid (DCTH) is toxic to rat liver and kidney mitochondria and is cytotoxic to isolated rat hepatocytes. The object of this investigation was to test the hypothesis that DCTH is bioactivated in vivo by the enzymes of mitochondrial fatty acid beta oxidation and that the observed mitochondrial dysfunction is a consequence of this bioactivation. DCTH was a potent nephrotoxin and hepatotoxin in Long-Evans rats, whereas the odd-chain-length analog 6,7-dichloro-5-thia-6-heptenoic acid was not toxic. DCTH produced morphological changes in renal proximal convoluted tubules and the liver. The increases in urinary protein, glucose and blood urea nitrogen concentrations were consistent with the renal lesions. Hepatic lesions were associated with an increase in plasma glutamate-pyruvate transaminase activity, a marked infiltration of lipid and depletion of glycogen concentrations. A pronounced decrease in plasma glucose concentrations was also observed. DCTH decreased fatty acid beta oxidation by 75% and 40% in liver and kidney mitochondria, respectively, isolated from DCTH-treated rats. In addition, medium-chain acyl-coenzyme A dehydrogenase activity was reduced by 25% in rat liver mitochondria incubated with DCTH. The data presented are consistent with the hypothesis that DCTH is bioactivated by the mitochondrial fatty acid beta-oxidation system and that mitochondria are a critical cellular target in DCTH-induced toxicity.     

Toxicity of oxygen to mitochondrial respiratory activity in hypothermically perfused canine kidneys

Respiratory activity of kidney cortex homogenates was measured after various periods of hypothermic pulsatile preservation of dog kidneys with cryoprecipitated plasma. There was a progressive loss of pyruvate plus malate-stimulated respiration (30 to 40% at 3 days and 70 to 80% at 5 days) and succinate-stimulated respiration (15 to 20% at 3 days and 50 to 60% at 5 days). Perfusion under conditions of low pO2 (30 to 40 mm Hg) or with inhibitors of the toxic effects of hyperbaric oxygen (CO2+ Mn2+) preserved homogenate respiratory activity better than with normal pO2 (150 mm Hg) or high pO2 (300 mm Hg). The results suggest that oxygen toxicity (lipid peroxidation) and the progressive loss of respiration in homogenates may be limiting factors in obtaining long-term preservation.