The effect of iron overload on the mitochondrial porphyrin level in the hexachlorobenzene induced experimental porphyria

Liver mitochondria isolated from rats treated with hexachlorobenzene plus iron, present a lower content of total porphyrin in respect to that of mitochondria from rats fed hexachlorobenzene alone. The in vitro mitochondrial porphyrin accumulation processes have been studied in mitochondria from iron loaded rats. It has been found that under these conditions the active porphyrin uptake process, which is driven by the K+ transmembrane gradient, is maximally inhibited in the presence of pentachlorophenol at a concentration similar to that found in vivo in the hexachlorobenzene experimental porphyria. By contrast the same degree of inhibition is presented by control mitochondria only in the presence of pentachlorophenol plus valinomycin, a condition which collapses the transmembrane K+ gradient. A strict correlation between porphyrin uptake and K+ concentration has been found in control as well as in iron treated mitochondria. A possible involvement of peroxidative reactions in the mitochondrial membranes has been proposed as a cause of the changes in the permeability properties of the mitochondrial membranes in the experimental chronic hepatic porphyria under conditions of iron overload.     

Structural and functional properties of rat liver mitochondria in hexachlorobenzene induced experimental porphyria

A possible link between changes in iron and porphyrin content in liver mitochondria, from rats treated with either hexachlorobenzene, iron, or hexachlorobenzene plus iron, as a function of treatment time and their structural-functional properties, has been investigated. Normal oxidative phosphorylation in mitochondria from rats treated with iron has been shown. By contrast a significant and constant uncoupling of the phosphorylative process, fully reversed by albumin, in mitochondria from rats treated with hexachlorobenzene and hexachlorobenzene plus iron has been presented. A possible involvement of pentachlorophenol in causing these abnormalities has been proposed.     

The effect of ferric iron complex on isolated rat liver mitochondria. I. Respiratory and electrochemical responses

Addition of iron(III)-gluconate complex to isolated rat liver mitochondria resulted in an increased iron content of mitochondria. Iron was accumulated through a relatively fast process (maximal uptake in less than 2 min incubation) by an energy-independent mechanism. The in vitro iron overload of mitochondria was associated with enhancement in the oxygen consumption, which was due to the induction of lipoperoxidative processes catalyzed by iron. It was found that a concentration of iron as low as 0.1 mM elicits a consistent production of malondialdehyde in mitochondria. Concomitant with the induction of lipoperoxidation a progressive fall in the mitochondrial membrane potential was observed. The occurrence of energy-consuming processes as a consequence of iron addition, and particularly the enhancement of endogenous Ca2+ cycling across the membrane, was suggested as the cause of the membrane potential drop.     

The effect of ferric iron complex on Ca2+ transport in isolated rat liver mitochondria

The in vitro effects of iron (III)-gluconate complex on the production of malondialdehyde and on the Ca2+ transport in isolated rat liver mitochondria were studied. A correlation between the concentration of iron added and the formation of malondialdehyde was found. The enhancement by iron of lipid peroxidative process in the mitochondrial membrane brought about the induction of Ca2+ release from mitochondria. Experimental evidence based on the membrane potential pattern of mitochondria pre-loaded with a low pulse of Ca2+ suggested that Ca2+ efflux was not due to a nonspecific increase in the inner membrane permeability, i.e. to a collapse of membrane potential, but rather to the activation of an apparently selective pathway for Ca2+ release.     

Transmembrane potential of liver mitochondria from hexachlorobenzene- and iron-treated rats

The respiratory parameters and the membrane of liver mitochondria from rats treated with either hexachlorobenzene, iron or hexachlorobenzene plus iron, to induce experimental porphyria, have been studied. Partial uncoupling of oxidative phosphorylation has been observed in mitochondria from hexachlorobenzen- and hexachlorobenzene plus iron-treated rats. Direct evidence has been pressented that this uncoupling is due to the action of pentochlorophenol endogenously formed by metabolism of hexachlorobenzene. No irreversible damage of mitochondrial membrane has been revealed under both these conditions. Normal oxidative phosphorylation has bee found in mitochondria from rats treated with iron alone. In contrast, they presented an anomalous membrane potential, fully restored by oligomycin. A possible involvement of lipid peroxidation process, induced by iron, in causing these abnormalities has been suggested.     

Effect of decreased ferrochelatase activity on iron and porphyrin content in mitochondria of mice with porphyria induced by griseofulvin

The content of iron and protoporphyrin in liver mitochondria from mice with porphyria induced by griseofulvin was measured. The amount of porphyrin was 0.0076 +/- 0.0043, 4.11 +/- 0.58 and 22.2 +/- 6.8 nmol/mg protein (n = 5) in mitochondria from control animals and animals treated with griseofulvin for 3 days and 4-5 weeks, respectively. The energy coupling of the mitochondria was greatly diminished after 4-5 weeks of treatment, and the ferrochelatase activity was inhibited 80-90%, compared to that of control animals. Mitochondrial preparations isolated by differential centrifugation were contaminated with iron-containing lysosomes which could be removed by Percoll density-gradient centrifugation. In purified mitochondrial preparations no change in the amount of non-heme iron was found after griseofulvin feeding, representing 3.36 +/- 0.15, 3.97 +/- 0.40 and 3.59 +/- 0.23 nmol/mg protein for control animals, 3 days- and 4-5 weeks-treated animals, respectively (n = 4). A mitochondrial iron pool previously identified in rat liver mitochondria and shown to be available for heme synthesis in vitro (Tanger?s, A. (1985) Biochim. Biophys. Acta 843, 199-207) was also present in mitochondria from mice. The magnitude of this iron pool, as well as its availability for heme synthesis, was not changed after treatment of the animals with griseofulvin. The fact that porphyrin, but not iron, accumulated in the mitochondria when ferrochelatase was inhibited is discussed with regard to our understanding of the process of heme synthesis and its regulation.     

Effect of decreased ferrochelatase activity on iron and porphyrin content in mitochondrai of mice with porphyria induced by griseofulvin

The content of iron and protoporphyrin in liver mitochondria from mice with porphyria induced by griseofulvin was measured. The amount of porphyrin was 0.0076 ± 0.0043, 4.11 ± 0.58 and 22.2 ± 6.8 nmol/mg protein (n = 5) in mitochondria from control animals and animals treated with griseofulvin for 3 days and 4–5 weeks, respectively. The energy coupling of the mitochondia was greatly diminished after 4–5 weeks of treatment, and the ferrochelatase activity was inhibited 80–90%, compared to that of control animals. Mitochondrial preparations isolated by differential centrifugation were contaminated with iron-containing lysosomes which could be removed by Percoll density-gradient centrifugation. In purified mitochondrial preparations no change in the amount of non-heme iron was found after griseofulvin feeding, representing 3.36±0.15, 3.97±0.40 and 3.59±0.23 nmol/mg protein for control animals, 3 days- and 4–5 weeks-treated animals, respectively (n = 4). A mitochondrial iron pool previously identified in rat liver mitochondria and shown to be available for heme synthesis in vitro (Tangerås, A. (1985)) Biochim. Biophys. Acta 843 199–207) was also present in mitochondria from mice. The magnitude of this iron pool, as well as its availability for heme synthesis, was not changed after treatment of the animals with griseofulvin. The fact that porphyrin, but not iron, accumulated in the mitochondria when ferrochelatase was inhibited is discussed with regard to our understanding of the process of heme synthesis and its regulation.     

Transmembrane potential of liver mitochondria from hexachlorobenzene-and iron-treated rats

The respiratory parameters and the membrane potential of liver mitochondria from rats treated with either hexachlorobenzene, iron or hexachlorobenzene plus iron, to induce experimental porphyria, have been studied. Partial uncoupling of oxidative phosphorylation has been observed in mitochondria from hexachlorobenzene- and hexachlorobenzene plus iron-treated rats. Direct evidence has been presented that this uncoupling is due to the action of pentachlorophenol endogenously formed by metabolism of hexachlorobenzene. No irreversible damage of mitochondria membrane has been revealed under both these conditions. Normal oxidative phosphorylation has been found in mitochondria from rats treated with iron alone. In contrast, they presented an anomalous membrane potential, fully restored by oligomycin. A possible involvement of lipid peroxidation process, induced by iron, in causing these abnormalities has been suggested.     

The effect of a ferric iron complex on isolated rat-liver mitochondria. III. Mechanistic aspects of iron-induced calcium efflux

Addition of iron(III)-gluconate complex to isolated rat liver mitochondria induced a net efflux of Ca2+ which was not inhibited by ruthenium red. This process resulted in the enhancement of Ca2+ cycling and a consequent membrane potential drop. Under these experimental conditions the content of mitochondrial glutathione did not appear to be critically modified, whereas an extensive oxidation of mitochondrial pyridine nucleotides was parallelly detected. Iron failed to induce appreciable changes in the oxidation level of pyridine nucleotides in mitochondria isolated from rats fed a selenium deficient diet, a condition in which mitochondrial glutathione peroxidase resulted inhibited by 80%. The iron-induced Ca2+ release in Se-deficient mitochondria appeared largely delayed and the membrane potential of these mitochondrial did not present gross alterations. Iron was also found to induce a transient increase in the mitochondrial cyanide-insensitive oxygen consumption. This effect was largely prevented by the addition of the hydrogen peroxide scavenger catalase. It was concluded that iron induced the activation of a specific Ca2+ efflux pathway via the oxidation of pyridine nucleotides due to the hydrogen peroxide metabolism by glutathione enzyme system.     

A proposed sequence of events for cadmium-induced mitochondrial impairment

Cadmium is a very important environmental toxicant, the cytotoxicity mechanism of which is likely to involve mitochondria as a target. In the present study we addressed the cause/effect relationship between the multiple cadmium-induced responses involving the mitochondrial energetic and oxidative status. Assays were performed with succinate-energized rat liver mitochondria incubated with 5 microM CdCl(2) for 0-25 min, in the absence or presence, respectively, of N-ethylmaleimide (NEM), butylhydroxytoluene (BHT), ruthenium red (RR), and cyclosporine A+ADP. A sequence of events accounting for cadmium-induced mitochondrial impairment is proposed, beginning with an apparent interaction of Cd(2+) with specific protein thiols in the mitochondrial membrane, which stimulates the cation's uptake via the Ca(2+) uniporter, and is followed by the onset of mitochondrial permeability transition (MPT); both effects dissipate the transmembrane electrical potential (Deltapsi), causing uncoupling, followed by an early depression of mitochondrial ATP levels. The respiratory chain subsequently undergoes inhibition, generating reactive oxygen species which together with iron mobilized by the cation, cause late, gradual mitochondrial membrane lipid peroxidation.     

Chromium(VI) interaction with plant and animal mitochondrial bioenergetics: a comparative study

The mechanism of Cr(VI)-induced toxicity in plants and animals has been assessed for mitochondrial bioenergetics and membrane damage in turnip root and rat liver mitochondria. By using succinate as the respiratory substrate, ADP/O and respiratory control ratio (RCR) were depressed as a function of Cr(VI) concentration. State 3 and uncoupled respiration were also depressed by Cr(VI). Rat mitochondria revealed a higher sensitivity to Cr(VI), as compared to turnip mitochondria. Rat mitochondrial state 4 respiration rate triplicated in contrast to negligible stimulation of turnip state 4 respiration. Chromium(VI) inhibited the activity of the NADH-ubiquinone oxidoreductase (complex I) from rat liver mitochondria and succinate-dehydrogenases (complex II) from plant and animal mitochondria. In rat liver mitochondria, complex I was more sensitive to Cr(VI) than complex II. The activity of cytochrome c oxidase (complex IV) was not sensitive to Cr(VI). Unique for plant mitochondria, exogenous NADH uncoupled respiration was unaffected by Cr(VI), indicating that the NADH dehydrogenase of the outer leaflet of the plant inner membrane, in addition to complexes III and IV, were insensitive to Cr(VI). The ATPase activity (complex V) was stimulated in rat liver mitochondria, but inhibited in turnip root mitochondria. In both, turnip and rat mitochondria, Cr(VI) depressed mitochondrial succinate-dependent transmembrane potential (Deltapsi) and phosphorylation efficiency, but it neither affected mitochondrial membrane permeabilization to protons (H+) nor induced membrane lipid peroxidation. However, Cr(VI) induced mitochondrial membrane permeabilization to K+, an effect that was more pronounced in turnip root than in rat liver mitochondria. In conclusion, Cr(VI)-induced perturbations of mitochondrial bioenergetics compromises energy-dependent biochemical processes and, therefore, may contribute to the basal mechanism underlying its toxic effects in plant and animal cells.     

Iron stimulation of free radical-mediated porphyrinogen oxidation by hepatic and renal mitochondria

Mitochondria from rat liver and kidney catalyze oxidation of uroporphyrinogen in the presence of NADH or succinate and the respiratory chain inhibitor, NaN3. The rate of porphyrinogen oxidation was substantially accelerated when iron as Fe+3-EDTA was added to reaction mixtures. This effect was partially attenuated by catalase, reduced glutathione (GSH) and other free radical scavengers. These results suggest that iron stimulates free radical-mediated porphyrinogen oxidation by tissue mitochondria under conditions of perturbed mitochondrial respiratory function. These observations suggest a mechanism by which iron could contribute to excess porphyrin excretion in various inherited or chemically-induced porphyrias.     

The effect of ferric iron complex on isolated rat liver mitochondria. II. Ion movements

It has been found that addition of iron(III)-gluconate complex to rat liver mitochondria disturbed the mitochondrial Ca2+ transport. Indirect evidence when the changes in the membrane potential during the transport of Ca2+ were followed, as well as direct evidence, when the fluxes of Ca2+ were monitored by a Ca2+-selective electrode, indicated that this iron complex induced an efflux of Ca2+ from liver mitochondria. The mechanisms by which iron induced Ca2+ release appeared to be linked to the induction of lipoperoxidation of mitochondrial membrane. The mitochondrial membrane, however, did not become irreversibly damaged under these conditions, as indicated by its complete repolarization. It was also shown that the induction by iron of lipoperoxidation brought about an efflux of K+ from mitochondria.     

Riboflavin-binding protein. Concentration and fractional saturation in chicken eggs as a function of dietary riboflavin.

In female rats with porphyria induced by hexachlorobenzene, the amounts of non-haem iron and porphyrins in liver mitochondrial fractions were increased almost 3-fold and greater than 500-fold respectively compared with that of untreated animals. A considerable fraction of both iron and porphyrins in this fraction was shown to be located in lysosomes. Thus mitochondrial preparations, which were further depleted of lysosomes by Percoll-density-gradient centrifugation, contained 2.78 +/- 0.75 and 2.99 +/- 0.49 nmol of non-haem iron/mg of protein when isolated from the liver of control rats and hexachlorobenzene-treated rats respectively. Mitochondria isolated from the liver of hexachlorobenzene-treated animals contained a pool of iron (about 1 nmol/mg of protein) that was available for haem synthesis in vitro. This pool is similar to that previously reported for mitochondria isolated from the liver of rats with normal haem synthesis. Hexachlorobenzene treatment, therefore, does not affect the iron status of the mitochondria.     

Perturbation in liver mitochondrial Ca2+ homeostasis in experimental iron overload: a possible factor in cell injury

The functional state of isolated mitochondria and specifically the integrity of the inner membrane, were investigated in the liver of rats made siderotic by dietary supplementation with carbonyl iron. The concentration of iron in the hepatic tissue increased progressively up to nearly 40 days and reached a steady-state level. When the iron content reached a threshold value (higher than 90 nmol/mg protein) the occurrence of in vivo lipid peroxidation in the mitochondrial membrane was detected. This process did not result in gross alterations in the mitochondrial membrane, as indicated by electron microscopy, phosphorylative capability and membrane potential measurements. On the contrary, the induction of lipoperoxidative reaction appeared to be associated with the activation of Ca2+ release from mitochondria. This was shown to occur as a consequence of rather subtle modifications in the inner membrane structure via a specific efflux route, which appeared to be linked to the oxidation level of mitochondrial pyridine nucleotides. The induction of this Ca2+ release from iron-treated mitochondria resulted in enhancement of Ca2+ cycling, a process which dissipates energy to reaccumulate into mitochondria the released Ca2+. The perturbation in mitochondrial Ca2+ homeostasis reported here may be a factor in the onset of cell damage in this experimental model of hepatic iron overload.     

Mechanism of iron transport to the site of heme synthesis inside yeast mitochondria.

The import of metals, iron in particular, into mitochondria is poorly understood. Iron in mitochondria is required for the biosynthesis of heme and various iron-sulfur proteins. We have developed an in vitro assay to follow the uptake of iron into isolated yeast mitochondria. By measuring the incorporation of iron into porphyrin by ferrochelatase in the matrix, we were able to define the mechanism of iron import. Iron uptake is driven energetically by a membrane potential across the inner membrane but does not require ATP. Only reduced iron is functional in generating heme. Iron cannot be preloaded in the mitochondrial matrix but rather has to be transported across the inner membrane simultaneously with the synthesis of heme, suggesting that ferrochelatase receives iron directly from the inner membrane. Transport of iron is inhibited by manganese but not by zinc, nickel, and copper ions, explaining why in vivo these ions are not incorporated into porphyrin. The inner membrane proteins Mmt1p and Mmt2p proposed to be involved in mitochondrial iron movement are not required for the supply of ferrochelatase with iron. Iron transport can be reconstituted efficiently in a membrane potential-dependent fashion in proteoliposomes that were formed from a detergent extract of mitochondria. Our biochemical analysis of iron import into yeast mitochondria provides the basis for the identification of components involved in transport.     

Studies on the effects of copper deficiency on rat liver mitochondria. I. Changes in mitochondrial composition

As part of an investigation of the lesions of copper (Cu) deficiency a study was undertaken of the copper, iron, cytochrome and fatty acid composition of liver mitochondria from Cu deficient and Cu-adequate control rats. Cu concentrations were significantly decreased in whole liver, liver mitochondria and in blood plasma. Total iron was significantly increased in whole liver but remained at the normal level in mitochondria. Cytochrome c oxidase (EC 1.9.3.1) and its component cytochromes a and a3 were significantly reduced in liver mitochondria from Cu-deficient rats, whereas there was no effect on the concentration of cytochromes b, c1 and c. Evidence from comparisons between cytochrome c oxidase activity and the amount of enzyme present, as assessed from the mitochondrial cytochrome a and a3 content, suggests that in addition to an absolute loss of enzyme, Cu-deficiency adversely affects the efficiency of the residual enzyme. Severe Cu deficiency had no effect on 'ageing' or 'swelling' properties of liver mitochondria, indicating no marked effects on fatty acid composition. Fatty acid analyses demonstrated a slight but significant increase in docosapentenoic acid (22:5) of Cu-deficient mitochondria, but since this represents a minor component there was no change observed in the 'unsaturation index'. It was concluded that, in contrast to previous reports, Cu deficiency of the severity reported did not have a deleterious effect on the integrity and permeability of the inner mitochondrial membrane as exemplified by any qualitative modification of fatty acid constitution per se.     

Altered subcellular and submitochondrial localization of CTP:phosphatidate cytidylyltransferase in the Morris 7777 hepatoma.

The subcellular and submitochondrial localization of CTP:phosphatidate cytidylyltransferase is altered in the Morris 7777 hepatoma. Mitochondria in this poorly differentiated tumor are the principal sites of CDP-diacylglycerol synthesis, in contrast to normal rat liver where the endoplasmic reticulum is most active. This enzyme activity was increased 17-fold in the outer mitochondrial membrane, and a 22% increase was noted in the inner mitochondrial membrane of the 7777 hepatoma as compared with the corresponding fractions from normal rat liver. Increased mitochondrial CTP:phosphatidate cytidylyltransferase was present in six other Morris hepatomas, but it was not found in fetal rat liver mitochondria, suggesting that rapid growth alone is not responsible for the difference. Evidence is presented which indicates that mitochondrial lipid degradation is similar in normal liver and the 7777 hepatoma, in vitro. The increased activity of CTP: phosphatidate cytidylytransferase is thought to be responsible in part for the moderately increased diphosphatidylglycerol content of 7777 hepatoma mitochondria.     

A study on the interaction of the amyloid fibrils of α-synuclein and hen egg white lysozyme with biological membranes

Alpha-synuclein (α-syn) aggregation and mitochondrial dysfunction are considered as two of the main factors associated with Parkinson's disease (PD). In the present investigation, the effectiveness of the amyloid fibrils obtained from α-syn with those of hen egg white lysozyme (HEWL), as disease-related and-unrelated proteins, to damage rat brain and rat liver mitochondria have been investigated. This was extended by looking at SH-SY5Y human neuroblastoma cells and erythrocytes, thereby investigating the significance of structural characteristics of amyloid fibrils related to their interactions with biomembranes obtained from various sources. Results presented clearly demonstrate substantial differences in the response of tested biomembranes to toxicity induced by α-syn/HEWL amyloid fibrils, highlighting a structure-function relationship. We found that fibrillar aggregates of α-syn, but not HEWL, caused a significant increase in mitochondrial ROS, loss of membrane potential, and mitochondrial swelling, in a dose-dependent manner. Toxicity was found to be more pronounced in brain mitochondria, as compared to liver mitochondria. For SH-SY5Y cells and erythrocytes, however, both α-syn and HEWL amyloid fibrils showed the capacity to induce toxicity. Taken together, these results may suggest selective toxicity of α-syn amyloid fibrils to mitochondria mediated likely by their direct interaction with the outer mitochondrial membrane, indicating a correlation between specific structural characteristics of α-syn fibrils and an organelle strongly implicated in PD pathology.     

Proton leak in hepatocytes and liver mitochondria from archosaurs (crocodiles) and allometric relationships for ectotherms

It has previously been shown that mitochondrial proton conductance decreases with increasing body mass in mammals and is lower in a 250-g lizard than the laboratory rat. To examine whether mitochondrial proton conductance is extremely low in very large reptiles, hepatocytes and mitochondria were prepared from saltwater crocodiles ( Crocodylus porosus) and freshwater crocodiles ( Crocodylus johnstoni). Respiration rates of hepatocytes and liver mitochondria were measured at 37 degrees C and compared with values obtained for rat or previously measured for other species. Respiration rates of hepatocytes from either species of crocodile were similar to those reported for lizards and approximately one fifth of the rates measured using cells from mammals (rat and sheep). Ten-to-thirty percent of crocodile hepatocyte respiration was used to drive mitochondrial proton leak, similar to the proportion in other species. Respiration rates of crocodile liver mitochondria were similar to those of mammalian species. Proton leak rate in isolated liver mitochondria was measured as a function of membrane potential. Contrary to our prediction, the mitochondrial proton conductance of liver mitochondria from crocodiles was greater than that of liver mitochondria from lizards and was similar to that of rats. The acyl composition of liver mitochondrial phospholipids from the crocodiles was more similar to that in mitochondria from rats than in mitochondria from lizards. The relatively high mitochondrial proton conductance was associated with a relatively small liver, which seems to be characteristic of crocodilians. Comparison of data from a number of diverse ectothermic species suggested that hepatocyte respiration rate may decrease with body mass, with an allometric exponent of about -0.2, similar to the exponent in mammalian hepatocytes. However, unlike mammals, liver mitochondrial proton conductance in ectotherms showed no allometric relationship with body size.