Oxidative stress as regulatory factor for fatty-acid-induced uncoupling involving liver mitochondrial ADP/ATP and aspartate/glutamate antiporters of old rats

Palmitate-induced uncoupling, which involves ADP/ATP and aspartate/glutamate antiporters, has been studied in liver mitochondria of old rats (22-26 months) under conditions of lipid peroxidation and inhibition of oxidative stress by antioxidants--thiourea, Trolox, and ionol. It has been shown that in liver mitochondria of old rats in the absence of antioxidants and under conditions of overproduction of conjugated dienes, the protonophoric uncoupling activity of palmitate is not suppressed by either carboxyatractylate or aspartate used separately. However, the combination of carboxyatractylate and aspartate decreased uncoupling activity of palmitate by 81%. In this case, palmitate-induced uncoupling is limited by a stage insensitive to both carboxyatractylate and aspartate. In the presence of antioxidants, the palmitate-induced protonophoric uncoupling activity is suppressed by either carboxyatractylate or aspartate used separately. Under these conditions, palmitate-induced uncoupling is limited by a stage sensitive to carboxyatractylate (ADP/ATP antiporter) or aspartate (aspartate/glutamate antiporter). In the absence of antioxidants, the uncoupling activity of palmitate is not suppressed by ADP either in the absence or in the presence of aspartate. However, in the presence of thiourea, Trolox, or ionol ADP decreased the uncoupling activity of palmitate by 38%. It is concluded that in liver mitochondria of old rats the development of oxidative stress in the presence of physiological substrates of ADP/ATP and aspartate/glutamate antiporters (ADP and aspartate) results in an increase of the protonophoric uncoupling activity of palmitate.     

Acetoacetate as regulator of palmitic acid-induced uncoupling involving liver mitochondrial ADP/ATP antiporter and aspartate/glutamate antiporter

The effect of acetoacetate on palmitate-induced uncoupling with the involvement of ADP/ATP antiporter and aspartate/glutamate antiporter has been studied in liver mitochondria. The incubation of mitochondria with acetoacetate during succinate oxidation in the presence of rotenone, oligomycin, and EGTA suppresses the accumulation of conjugated dienes. This is considered as a display of antioxidant effect of acetoacetate. Under these conditions, acetoacetate does not influence the respiration of mitochondria in the absence or presence of palmitate but eliminates the ability of carboxyatractylate or aspartate separately to suppress the uncoupling effect of this fatty acid. The action of acetoacetate is eliminated by β-hydroxybutyrate or thiourea, but not by the antioxidant Trolox. In the absence of acetoacetate, the palmitate-induced uncoupling is limited by a stage sensitive to carboxyatractylate (ADP/ATP antiporter) or aspartate (aspartate/glutamate antiporter); in its presence, it is limited by a stage insensitive to the effect of these agents. In the presence of Trolox, ADP suppresses the uncoupling action of palmitate to the same degree as carboxyatractylate. Under these conditions, acetoacetate eliminates the recoupling effects of ADP and aspartate, including their joint action. This effect of acetoacetate is eliminated by β-hydroxybutyrate or thiourea. It is supposed that the stimulating effect of acetoacetate is caused both by increase in the rate of transfer of fatty acid anion from the inner monolayer of the membrane to the outer one, which involves the ADP/ATP antiporter and aspartate/glutamate antiporter, and by elimination of the ability of ADP to inhibit this transport. Under conditions of excessive production of reactive oxygen species in mitochondria at a high membrane potential and in the presence of small amounts of fatty acids, such effect of acetoacetate can be considered as one of the mechanisms of antioxidant protection.     

Involvement of phosphate carrier as a part of complex with ADP/ATP and aspartate/glutamate antiporters in palmitic acid-induced uncoupling in liver mitochondria

In liver mitochondria, the phosphate carrier is involved in protonophoric uncoupling effect of fatty acids together with ADP/ATP and aspartate/glutamate antiporters (Samartsev et al. 2003. Biochemistry (Moscow). 68, 618–629). Liver mitochondria depleted of endogenous oxidation substrates (exhausted mitochondria) have been used in the present work. In these mitochondria, like in the intact liver mitochondria, the specific inhibitor of ADP/ATP antiporter (carboxyatractylate) and the substrate of aspartate/glutamate antiporter (aspartate) suppress the uncoupling activity of palmitic acid. It is shown that in exhausted mitochondria the substrate of phosphate carrier (inorganic phosphate) and its nonspecific inhibitor mersalyl partially suppress palmitic acid-induced uncoupling due to decrease in the component of uncoupling activity sensitive to carboxyatractylate and aspartate. In the presence of inorganic phosphate or mersalyl, carboxyatractylate and aspartate added separately subsequent to palmitic acid do not suppress its uncoupling activity. They are effective only when added jointly. In the presence of thiourea or pyruvate, such effects of inorganic phosphate and mersalyl are not observed. It is supposed that in the presence of inorganic phosphate or mersalyl and under the condition of oxidation of critical SH-groups in mitochondria, the phosphate carrier, ADP/ATP antiporter, and aspartate/glutamate antiporter are involved in uncoupling function together with the general fatty acid pool as an uncoupling complex. The role of phosphate carrier in this complex may consist in facilitation of lateral transfer of the fatty acid molecules from one antiporter to another.     

Synthesis and antioxidative activity of metalloporphyrins bearing 2,6-di-tert-butylphenol pendants

The novel metalloporphyrins (M = HH, Fe, Mn, Co, Cu, Zn) bearing 2,6-di-tert-butylphenol pendants as antioxidant substituents, and a long chain hydrocarbon palmitoyl group have been synthesized. The oxidation of compounds by PbO₂ leads to the formation of the corresponding 2,6-di-tert-butylphenoxyl radicals studied by EPR. The activity of porphyrins in lipid peroxidation has been examined using (1) in vitro lipid peroxidation induced by tert-butylhydroperoxide in respiring rat liver mitochondria, (2) in vitro lipid peroxidation in liver homogenates of Wistar strain rats, and (3) a model process of peroxidation of (Z)-octadec-9-enic (oleic) acid as a structural fragment of lipids. The activity of these compounds depends dramatically on the nature of metal and might be changed from antioxidative (M = HH, Mn, Cu, Zn) to indifferent (M = Co), and to pro-oxidative one (M = Fe). The anti- or pro-oxidative action of these compounds may be derived from the concurrence between the involvement of 2,6-di-tert-butylphenol pendants acting as radical scavengers and redox active metal center promoting oxidation processes. The results of this study suggest that the polytopic compounds combining in one molecule 2,6-di-tert-butylphenol pendants, metalloporphyrin moiety, and a palmitoyl group, are membrane active compounds and might be studied in an effort to find novel pharmaceutical agents.     

Participation of ADP/ATP- and Aspartate/Glutamate Antiporters in the Uncoupling Action of Fatty Acids in Liver Mitochondria of the Frog Rana temporaria

Data are presented on molecular mechanisms of uncoupling of oxidative phosphorylation by fatty acids (laurate) in liver mitochondria of one of the poikilothermal animals, the frog Rana temporaria. It has been shown that the uncoupling action of laurate in frog liver mitochondria, like in those of mammals, occurs with participation of protein carriers of anions of the inner mitochondrial membrane, ADP/ATP- and aspartate/glutamate antiporters. At the same time, in frog liver mitochondria the uncoupling activity of laurate is lower than in liver mitochondria of mammals (white mice). Seasonal differences in the laurate uncoupling activity in frog liver mitochondria are revealed: it is much lower in April, than in January, the season of metabolic depression. This difference is due to that in January the degree of participation of the aspartate/glutamate antiporter in the uncoupling is considerably decreased.     

The ATP/ADP-antiporter is involved in the uncoupling effect of fatty acids on mitochondria

The ATP/ADP-antiporter inhibitors and the substrate ADP suppress the uncoupling effect induced by low (10-20 microM) concentrations of palmitate in mitochondria from skeletal muscle and liver. The inhibitors and ADP are found to (a) inhibit the palmitate-stimulated respiration in the controlled state and (b) increase the membrane potential lowered by palmitate. The degree of efficiency decreases in the order: carboxyatractylate (CAtr) greater than ADP greater than bongkrekic acid, atractylate. GDP is ineffective, Mg.ADP is of much smaller effect, whereas ATP is effective at much higher concentration than is ADP. Inhibitor concentrations, which maximally suppress the palmitate-stimulated respiration, correspond to those needed for arresting the state 3 respiration. The extent of the CAtr-sensitive stimulation of respiration by palmitate has been found to decrease with an increase in palmitate concentration. Stimulation of the controlled respiration by p-trifluoromethoxycarbonylcyanide phenylhydrozone (FCCP) and gramicidin D at any concentrations of these uncouplers is CAtr-insensitive, whereas that caused by a low concentrations of 2,4-dinitrophenol and dodecyl sulfate is inhibited by CAtr. The above effect of palmitate develops immediately after addition of the fatty acid. It is resistant to EGTA as well as to inhibitors of phospholipase (nupercain) and of lipid peroxidation (ionol). Moreover, palmitate accelerates spontaneous release of the respiratory control, developing in rat liver mitochondria under certain conditions. This effect takes several minutes, being sensitive to EGTA, nupercain and ionol. Like the fast uncoupling, this slow effect is inhibited by ADP but CAtr and atractylate are stimulatory rather than inhibitory. In artificial planar phospholipid membrane, palmitate does not increase the membrane conductance, FCCP increases it strongly and dinitrophenol only slightly. In cytochrome oxidase proteoliposomes, FCCP, gramicidin and dinitrophenol (less effectively) lower, whereas palmitate enhances the cytochrome-oxidase-generated membrane potential. In this system, monensin substitutes for palmitate. It is concluded that the ATP/ADP antiporter is somehow involved in the uncoupling effect caused by low concentrations of palmitate and, partially, of dinitrophenol, whereas uncoupling produced by FCCP and gramicidin is due to their action on the phospholipid part of the mitochondrial membrane. A possible mechanism of this effect is discussed.     

Effect of ethanol on the palmitate-induced uncoupling of oxidative phosphorylation in liver mitochondria

The effect of ethanol on the uncoupling activity of palmitate and recoupling activities of carboxyatractylate and glutamate was studied in liver mitochondria at various Mg²⁺ concentrations and medium pH values (7.0, 7.4, and 7.8). Ethanol taken at concentration of 0.25 M had no effect on the uncoupling activity of palmitic acid in the presence of 2 mM MgCl₂ and decreased the recoupling effects of carboxyatractylate and glutamate added to mitochondria either just before or after the fatty acid. However, ethanol did not modify the overall recoupling effect of carboxyatractylate and glutamate taken in combination. The effect of ethanol decreased as medium pH was decreased to 7.0. Elevated concentration of Mg²⁺ (up to 8 mM) inhibits the uncoupling effect of palmitate. Ethanol eliminates substantially the recoupling effect of Mg²⁺ under these conditions, but does not influence the recoupling effects of carboxyatractylate and glutamate. It is inferred that ADP/ATP and aspartate/glutamate antiporters are involved in uncoupling function as single uncoupling complex with the common fatty acid pool. Fatty acid molecules gain the ability to migrate under the action of ethanol: from ADP/ATP antiporter to aspartate/glutamate antiporter on addition of carboxyatractylate and in opposite direction on addition of glutamate. Possible mechanisms of fatty acid translocation from one transporter to another are discussed.     

tert–butylhydroperoxide—Induced toxicity in isolated hepatocytes: Contribution of thiol oxidation and lipid peroxidation

Incubation of isolated rat hepatocytes with tert-butylhydroperoxide resulted in marked cytotoxicity preceded by intracellular glutathione depletion and extensive lipid peroxidation. Addition of antioxidants delayed, but did not prevent, this toxicity. A significant decrease in protein-free sulfhydryl groups also, occurred in the presence of tert-butylhydroperoxide; direct oxidation of protein thiols and mixed disulfide formation with glutathione were responsible for this decrease. The involvement of protein thiol depletion in tert-butylhydroperoxide–induced cytotoxicity is suggested by our observation that administration of dithiothreitol, which caused re-reduction of the oxidized sulfhydryl groups and mixed disulfides, efficiently protected the cells from toxicity. Moreover, depletion of intracellular glutathione by pretreatment of the hepatocytes with diethyl maleate accelerated and enhanced the depletion of protein thiols induced by tert-butylhydroperoxide and potentiated cell toxicity even in the absence of lipid peroxidation.     

Features of the Uncoupling Effect of Fatty Acids in Liver Mitochondria of Mammals with Different Body Weight

In the presence of oligomycin, EGTA, and magnesium ions, the protonophore uncoupling activity of palmitate (V(Pal)) is determined as the ratio of the acceleration of respiration with palmitate to its concentration. Under these conditions, V(Pal) in liver mitochondria of one-month-old rats with the body weight of 50 g is 1.46-fold higher than in liver mitochondria of adult rats with the body weight of 250 g, whereas the uncoupling activity of FCCP does not depend on the age of the animals. The difference in V(Pal) is mainly due to its component insensitive to carboxyatractylate and glutamate (V(Ins)). This value is 2.9-fold higher in mitochondria of one-month-old rats than in those of adult rats. The protonophore activity of palmitate is similar in liver mitochondria of four-day-old and adult rats. In liver mitochondria of adult mammals (mouse, rat, guinea pig, rabbit), V(Pal) decreases with increase in the body weight of the animals. In double logarithmic coordinates, the dependence of the V(Pal) value on the body weight is linear with slope angle tangent of -0.18. The V(Pal) value is mainly contributed by its component V(Ins). In the presence of calcium ions, palmitate induces the nonspecific permeability of the inner membrane of liver mitochondria (pore opening). This Ca2+-dependent uncoupling effect of palmitate is less pronounced in mitochondria of one-month-old rats than in those of adult rats. In mitochondria of adult animals (mice, rats, and guinea pigs), the Ca2+-dependent uncoupling activity of palmitate is virtually the same. It is concluded that the protonophore uncoupling effect of palmitate in liver mitochondria of mammals, unlike its Ca2+-dependent effect, is associated with thermogenesis at rest and also with production of additional heat on cooling of the animals.     

Simulation of the uncoupling activity of fatty acids with the participation of ADP/ATP and aspartate/glutamate antiporters in liver mitochondria

It has been found that the protonophoric specific uncoupling activity of palmitic acid in rat liver mitochondria does not change as its concentration increases from 5 to 40 μM. Under these conditions, the component of the specific uncoupling activity that describes the participation in uncoupling of the ADP/ATP antiporter (sensitive to carboxyatractylate) increases, and the component of specific uncoupling activity that characterizes the participation in the uncoupling of the aspartate/glutamate antiporter (sensitive to glutamate) decreases by the same value. A kinetic model of the fatty acid-induced uncoupling activity with the participation of ADP/ATP and aspartate/glutamate antiporters has been developed. According to the model, these carriers can exist in two forms: active, i.e., participating in the uncoupling, and inactive. The interaction of a fatty acid with the regulator site of the ADP/ATP antiporter translates it from the inactive to the active form, while the interaction of a fatty acid with the regulator site of the aspartate/glutamate antiporter, on the contrary, translates it from the active form to inactive. The velocity of transport of a fatty acid anion by the antiporter from the internal monolayer of the inner membrane to the external monolayer is proportional to the product of the concentration of the fatty acid and the active form of this carrier. A good conformity of the model to experimentally obtained data is shown provided that (a) ADP/ATP and aspartate/glutamate antiporters, being completely in active state, transfer fatty acid anions with the same velocity; (b) the equilibrium dissociation constants of a complex of the carrier with the fatty acid in these antiporters are equal.     

Free fatty acids as inducers and regulators of uncoupling of oxidative phosphorylation in liver mitochondria with participation of ADP/ATP- and aspartate/glutamate-antiporter

In liver mitochondria fatty acids act as protonophoric uncouplers mainly with participation of internal membrane protein carriers — ADP/ATP and aspartate/glutamate antiporters. In this study the values of recoupling effects of carboxyatractylate and glutamate (or aspartate) were used to assess the degree of participation of ADP/ATP and aspartate/glutamate antiporters in uncoupling activity of fatty acids. These values were determined from the ability of these recoupling agents to suppress the respiration stimulated by fatty acids and to raise the membrane potential reduced by fatty acids. Increase in palmitic and lauric acid concentration was shown to increase the degree of participation of ADP/ATP antiporter and to decrease the degree of participation of aspartate/glutamate antiporter in uncoupling to the same extent. These data suggest that fatty acids are not only inducers of uncoupling of oxidative phosphorylation, but that they also act the regulators of this process. The linear dependence of carboxyatractylate and glutamate recoupling effects ratio on palmitic and lauric acids concentration was established. Comparison of the effects of fatty acids (palmitic, myristic, lauric, capric, and caprylic having 16, 14, 12, 10, and 8 carbon atoms, respectively) has shown that, as the hydrophobicity of fatty acids decreases, the effectiveness decreases to a greater degree than the respective values of their specific uncoupling activity. The action of fatty acids as regulators of uncoupling is supposed to consist of activation of transport of their anions from the internal to the external monolayer of the internal membrane with participation of ADP/ATP antiporter and, at the same time, in inhibition of this process with the participation of aspartate/glutamate antiporter.     

Simulation of the uncoupling activity of fatty acids with the participation of ADP/ATP and aspartate/glutamate antiporters in liver mitochondria

It has been found that the protonophoric specific uncoupling activity of palmitic acid in rat liver mitochondria does not change as its concentration increases from 5 to 40 microM. Under these conditions, the component of the specific uncoupling activity, which describes the participation in uncoupling of the ADP/ATP antiporter (sensitive to carboxyatractylate), increases, and the component of specific uncoupling activity, which characterizes the participation in the uncoupling of the aspartate/glutamate antiporter (sensitive to glutamate), decreases by the same value. A kinetic model of the fatty acid-induced uncoupling activity with the participation of ADP/ATP and aspartate/glutamate antiporters has been developed. According to the model, these carriers can exist in two forms: an active, i.e., participating in the uncoupling, and an inactive. The interaction of a fatty acid with the regulator site of the ADP/ATP antiporter translates it from the inactive to the active form, while the interaction of a fatty acid with the regulator site of the aspartate/glutamate antiporter, on the contrary, translates it from the active form to inactive. The velocity of transport of a fatty acid anion by the antiporter from the internal monolayer of the internal membrane to the external monolayer is proportional to the product of the concentration of the fatty acid and the active form of this carrier. A good conformity of the model to experimentally obtained data is shown provided that (a) ADP/ATP and aspartate/glutamate antiporters, being completely in an active state, transfer fatty acid anions with the same velocity; (b) the equilibrium dissociation constants of a complex of the carrier with the fatty acid in these antiporters are equal.     

Radiosensitive antioxidant membrane-bound factors in rat liver microsomes: I. The roles of glutathione and vitamin E

In vitro lipid peroxidation initiated by NADPH/ADP/Fe3+ reveals an alteration of rat liver microsomal antioxidant factors at day D+4 after whole-body gamma irradiation (8Gy). This alteration is partly reversed by GSH, and more efficiently by Trolox C, a water-soluble analog of vitamin E. This reversion by Trolox C, together with the observed 50% decrease in vitamin E content in microsomes of irradiated rats as compared to those of control animals, indicate that Trolox C acts as a free-radical scavenger like and in place of vitamin E. The antioxidant action of Trolox C is not improved in the presence of GSH, which suggests that the former acts earlier than the latter on the autoxidative free-radical chain reactions. Neither GSH, nor Trolox C, nor both antioxidants totally inhibit in vitro lipid peroxidation, which appeals attention on the possible role of extra-microsomal antioxidant factors, especially cytosolic ones.     

Changes in intramitochondrial adenine nucleotides in blowfly flight-muscle mitochondria

1. With freshly isolated blowfly mitochondria 38% of the intramitochondrial adenine nucleotide was present as AMP. 2. On incubation with oxidizable substrates the AMP and ADP concentrations fell and that of ATP rose; with pyruvate together with proline the ATP concentration reached its maximum value at 6min; with glycerol phosphate the phosphorylation of endogenous nucleotide was more rapid. 3. Addition of the uncoupling agent carbonyl cyanide phenylhydrazone caused a rapid fall of ATP and a parallel rise in ADP, then ADP was converted into AMP. 4. This was in contrast with rat liver mitochondria endogenous AMP concentrations, which were always lower than those of blowfly mitochondria and changed little under different metabolic conditions. 5. Evidence is presented that adenylate kinase (EC 2.7.4.3) has a dual distribution in blowfly mitochondria, a part being located in the matrix space and a part in the space between the outer and inner mitochondrial membranes, as in liver and other mitochondria. 6. The possible regulatory role of changing AMP concentrations in the mitochondrial matrix was investigated. Partially purified pyruvate carboxylase (EC 6.4.1.1) and citrate synthase (EC 4.1.3.7) were inhibited 30% by 2mm-AMP, whereas pyruvate dehydrogenase (EC 1.2.4.1) was unaffected. 7. AMP activated the NAD(+)-linked isocitrate dehydrogenase (EC 1.1.1.41) activity of blowfly mitochondria in the absence of ADP, but in the presence of ADP, AMP caused inhibition. 8. It is suggested that AMP may exert a controlling effect on the oxidative activity of blowfly mitochondria.     

Effect of the cationic detergent CTAB on the involvement of ADP/ATP antiporter and aspartate/glutamate antiporter in fatty acid-induced uncoupling of liver mitochondria

The influence of the positively charged amphiphilic compound cetyltrimethyl ammonium bromide (CTAB) on palmitate- and laurate-induced uncoupling and on carboxyatractylate and glutamate recoupling effects in liver mitochondria have been studied. CTAB (40 M) in the presence of 3 mM MgCl₂ had little (if any) effect on the palmitic acid-stimulated respiration of mitochondria; the glutamate recoupling effect increased, and the carboxyatractylate recoupling effect decreased to the same degree with the combined effect (about 80%) remaining unchanged. Thus, CTAB decreases the ADP/ATP antiporter involvement and increases to the same extent the aspartate/glutamate antiporter involvement in the fatty acid-induced uncoupling. The carboxyatractylate and glutamate recoupling effects were less pH dependent in the presence of CTAB than in its absence. These data could be interpreted with the assumption that fatty acid anions are more accessible to the ADP/ATP antiporter and their neutral forms are more accessible to the aspartate/glutamate antiporter, and that CTAB changes the relative anion carrier involvement in the fatty acid-induced uncoupling as it forms neutral complexes with fatty acid anions.     

Inhibition of Nitric-Oxide-Mediated Apoptosis in Jurkat Leukemia Cells despite Cytochrome c Release

We have recently shown that nitric-oxide (NO)-induced apoptosis in Jurkat human leukemia cells requires degradation of mitochondria phospholipid cardiolipin, cytochrome c release, and activation of caspase-9 and caspase-3. Moreover, an inhibitor of lipid peroxidation, Trolox, suppressed apoptosis in Jurkat cells induced by NO donor glycerol trinitrate. Here we demonstrate that this antiapoptotic effect of Trolox occurred despite massive release of the mitochondrial protein cytochrome c into the cytosol and mitochondrial damage. Incubation with Trolox caused a profound reduction of intracellular ATP concentration in Jurkat cells treated by NO. Trolox prevented cardiolipin degradation and caused its accumulation in Jurkat cells. Furthermore, Trolox markedly downregulated the NO-mediated activation of caspase-9 and caspase-3. Caspase-9 is known to be activated by released cytochrome c and together with caspase-3 is considered the most proximal to mitochondria. Our results suggest that the targets of the antiapoptotic effect of Trolox are located downstream of the mitochondria and that caspase activation and subsequent apoptosis could be blocked even in the presence of cytochrome c released from the mitochondria.     

The effect of melatonin on free radical homeostasis in rat tissues at thyrotoxicosis

Experimental thyrotoxicosis in rats is accompanied by the increase of serum alanine aminotransferase (AlAT), aspartate aminotransferase (AsAT), creatine phosphokinase-MB (CPK-MB) activities and the content of primary products of lipid peroxidation, conjugated dienes, in liver, heart and blood. This suggests impairments in functioning of these organs, which accompany intensification of free radical processes. Melatonin administration resulted in the decrease of AlAT, AsAT, CPK-MB and conjugated dienes; this indicates positive effect of melatonin in this pathology. Thyrotoxicosis is accompanied by the increase of catalase activity in rat liver, heart and serum. Exogenous melatonin decreased specific activity of serum and heart catalase by 22 and 43%, respectively, compared with rats subjected to hyperthyroidism. However, there was insignificant increase in specific activity of liver catalase (by ～15%). Melatonin administration caused a decrease of α-tocopherol content increased in rat tissues under conditions of hyperthyroidism. Thus, exogenous melatonin is capable to reduce intensity of lipid peroxidation in hyperthyroidism and to act as an adaptogen, regulating free radical homeostasis in response to action of pathogenic factors on organism that is associated by concomitant reduction of mobilization of components of the antioxidant system.     

Lipid peroxidation in mitochondria and microsomes from adult and fetal rat tissues

Pregnant female Wistar rats that received a control (100 ppm Zn) or a Zn-deficient diet (1.5 ppm Zn) from d 0 to 21, or nonpregnant normally fed female rats without or with five daily oral doses of 300 mg/kg salicylic acid were used for the experiments. In isolated mitochondria or microsomes from various maternal and fetal tissues, lipid peroxidation was determined as malondialdehyde formation measured by means of the thiobarbiturate method. Zn deficiency increased lipid peroxidation in mitochondria and microsomes from maternal and fetal liver, maternal kidney, maternal lung microsomes, and fetal lung mitochondria. Lipid peroxidation in fetal microsomes was very low. Zn deficiency produced a further reduction of lipid peroxidation in fetal liver microsomes. Salicylate increased lipid peroxidation in liver mitochondria and microsomes after addition in vitro and after application in vivo. The increase of lipid peroxidation by salicylate may be caused by two mechanisms: an increased cellular Fe uptake that, in turn, can increase lipid peroxidation and chelating Fe, in analogy to the effect of ADP in lipid peroxidation. The latter effect of salicylate is particularly expressed at increased Fe content.     

Enterocyte mitochondrial dysfunction due to oxidative stress

The endogenous production of H2O2 in isolated rat intestinal mitochondria and oxidant induced damage to mitochondria were examined. There was an appreciable amount of H2O2 production in presence of succinate, glutamate and pyruvate, while the presence of rotenone with succinate further increased production. Superoxide generated by the X-XO system induced membrane permeability transition (MPT), calcium influx, lipid peroxidation and changes in membrane fluidity in mitochondria. A decreased mitochondrial ATPase activity and uncoupling of respiration was also observed. Spermine inhibited swelling induced by X-XO and also blocked the calcium influx and reversed the membrane fluidity changes.