Cytochrome P450 inactivation in microsomal membranes damaged by Fe2+-ascorbate-dependent lipid peroxidation

It has been established that Fe2+-ascorbate-dependent lipid peroxidation in rat liver microsome membranes is followed by the decrease of microsome cytochrome P450 content and the increase of the reduced haemoprotein inactivation rate. These changes are proportional to the amount of lipid peroxidation products (malonic dialdehyde) accumulating in the membranes.     

Fe2+-supported in vivo lipid peroxidation induced by compounds undergoing redox cycling

Treatment of male mice with the redox cycling compounds nitrofurantoin, paraquat, diquat or menadione failed to elicit in vivo lipid peroxidation as evidenced by ethane exhalation. The first three led to an enhanced ethane production, however, when the animals were pretreated with a low dose of Fe2+. While GSH-depletion by phorone pretreatment alone had no influence on the in vivo lipid peroxidation as evidenced by ethane expiration in the presence of either compound, the combined treatment with phorone, Fe2+ and nitrofurantoin, paraquat or diquat led to a further enhancement of ethane exhalation. These results indicate that redox cycling compounds do not initiate lipid peroxidation by themselves, but are well capable of stimulating the iron-induced LPO.     

Initiation of nonenzymatic lipid peroxidation in a Fe2+-ascorbate-linolenate system

Using spin trapping method there were discovered and identified the radicals of linolenic acid formed when initiating its peroxidation by the system Fe2+-ascorbate. Mechanism of formation of linolenic acid radicals and their role in initiation of peroxidation were studied. A scheme of reactions of peroxidation initiation in the system Fe2+-ascorbate. linolenic acid is proposed.     

Two phases in development of chemiluminescence during lipid peroxidation induced by Fe2+ ions

After cessation of the stirring of reagents in the egg yolk lipoprotein suspension where lipid peroxidation had been initiated by ferrous ions a drop of chemiluminescence (CL) was observed after a lag period, the duration of which increased in the course of stationary-state CL development. The CL intensity was but slightly dependent on oxygen pressure in the gas phase above incubation mixture within oxygen partial pressure limits 760-40 mm Hg. These data suggest that the CL quantum yield is growing during lipid peroxidation development as a result of the formation in the system of some compounds amplifying the CL intensity and/or development of a new CL reaction whose rate depends purely on oxygen and whose photon emitters are different.     

Cumene peroxide and Fe2+-ascorbate-induced lipid peroxidation and effect of phosphoglucose isomerase

Malondialdehyde (MDA) is one of cytotoxic aldehydes produced in cells as a result of lipid peroxidation and further MDA metabolism in cytoplasm is not known. In our experiments the liver fraction 10,000 g containing phosphoglucose isomerase and enzymes of the glyoxalase system was used and obtained experimental data shows that in this fraction there is an aggregate of reactions taking place both in membranes (lipid peroxidation) and outside membranes. MDA accumulation is relatively slow because MDA is a substrate of aldehyde isomerase (MDA ↔ methylglyoxal). The well known enzyme phosphoglucose isomerase acts as an aldehyde isomerase (Michaelis constant for this enzyme Km = 133 ± 8 μM). MDA conversion to methylglyoxal and further to neutral product D-lactate (with GSH as a cofactor) occurs in cytoplasm and D-lactate should be regarded as the end product of two different parametabolic reactions: lipid peroxidation or protein glycation.     

Tryptophan fluorescence of erythrocyte ghosts following irradiation and Fe2+ initiation of lipid peroxidation

It was shown that under the effect of Fe2+-initiated lipid peroxidation and ionizing radiation tryptophan fluorescence parameters (i.e. intensity and polarization) were subjected to similar changes. Shortly (15 min) after irradiation no changes were observed in the level of products reacting with thiobarbituric acid. It is concluded that the process and products of lipid peroxidation do not markedly contribute to the postirradiation alteration of tryptophan fluorescence. At the same time additional postirradiation damages to proteins can be attributed to activation of lipid peroxidation.     

Fluorescence emitted from microsomal membranes by lipid peroxidation

The fluorescence emitted from rat liver microsomal membranes which had undergone enzymatic and nonenzymatic lipid peroxidation was detected directly. This fluorescence produced in peroxidized membranes increased progressively with peroxidation reaction time, and the fluorescent substances produced were retained in the membranes without being released into the aqueous phase. Extracts of the peroxidized membranes with organic solvents (chloroform/methanol) emitted fluorescence which was also dependent on the peroxidation reaction time. The generation profiles of fluorescence emitted from both the peroxidized membranes and their extracted membrane lipids differed essentially from that of thiobarbituric acid-reactive substances which reached a plateau at a relatively early stage of peroxidation reaction. These results indicate that lipid peroxidation induces stepwise chemical and physical changes in membranes and that the fluorescence from peroxidized membranes will be useful in studying such changes occurring in biological membranes.     

Inhibition by the protein ceruloplasmin of lipid peroxidation stimulated by an Fe3+-ADP-adriamycin complex

Self-reduction of an Fe3+-ADP-adriamycin complex under anaerobic conditions and reduction of ferricytochrome c by the complex under aerobic conditions were strongly inhibited by ceruloplasmin, but not by superoxide dismutase or albumin at the same protein concentration. Ceruloplasmin, a protein with ferroxidase activity, is able to catalyse oxidation of Fe2+ to the ferric state. The inhibitory activity of ceruloplasmin towards reactions stimulated by the complex suggests that Fe2+ is formed during the self-reduction process. As expected, the Fe3+-ADP-adriamycin complex stimulated lipid peroxidation in which the Fe2+ moiety was implicated. This stimulation was again effectively prevented by ceruloplasmin but not by superoxide dismutase.     

Inhibition of microsomal lipid peroxidation by cytosolic protein in presence of ADP and high concentration of Fe2+

Microsomal lipid peroxidation induced by NADPH, but not by ascorbate, was found to be inhibited by liver cytosol. This inhibition was not dependent on glutathione and was enhanced by ADP in presence of Fe2+ at a concentration of 50 microM or higher. ATP was also effective, but not AMP or cyclic AMP. The cytosolic factor appeared to be a protein as it was heat-labile (greater than 70 degrees C), was non-dialyzable and was precipitated by ammonium sulfate and acetone. It was stable for several months in frozen state and also when heated at 50 degrees C for 10 min. The inhibition by the cytosolic protein was obtained by producing a lag in the activity of lipid peroxidation and was reversed by ceruloplasmin but not by catalase, cytochrome c, hemoglobin or superoxide dismutase. This inhibitory effect by cytosol was limited to formation of lipid peroxides whereas oxygen uptake and NADPH oxidation remained unaffected. Regulation of lipid peroxidation by nucleotide-Fe complexes and cytosolic proteins is indicated by these studies.     

The role of Fe2+-induced lipid peroxidation in the initiation of the mitochondrial permeability transition

Iron and iron complexes stimulate lipid peroxidation and formation of malondialdehyde (MDA). We have studied the effects of Fe2+ and ascorbate on mitochondrial permeability transition induced by phosphate and Ca2+. Iron is necessary for detectable MDA formation, but only Ca2+ and phosphate are necessary for the induction of membrane potential loss (Deltapsi) and Ca2+ release. Keeping the iron at a constant concentration and varying the Ca2+ level changed the mitochondrial Ca2+ retention times, but not the amount of MDA formation. The antioxidant butylated hydroxytoluene at low concentrations prevented MDA formation, but not mitochondrial Ca2+ release. Preincubation of mitochondria with Fe2+ decreased Ca2+ retention time in a concentration-dependent manner and facilitated Ca2+-stimulated MDA accumulation. Thus, Ca2+ phosphate-induced mitochondrial permeability transition (MPT) can be separated mechanistically from MDA accumulation. Lipid peroxidation products do not appear to participate in the initial phase of the permeability transition, but sensitize mitochondria toward MPT.     

Solubilization of adenylate cyclase of brain membranes by lipid peroxidation

Adenylate cyclase in the membrane fractions of bovine and rat brains, but not in rat liver plasma membranes, was solubilized by treatment with Fe²⁺ (10 μM) plus dithiothreitol (5 mM). Solubilization of the enzyme by these agents was completely prevented by simultaneous addition of N,N′-diphenyl-p-phenylenediamine (DPPD), an inhibitor of lipid peroxidation. Ascorbic acid also solubilized the enzyme from the brain membranes. Lipid peroxidation of the brain membranes was characterized by a selective loss of phosphatidylethanolamine. Solubilization of membrane-bound enzymes by Fe²⁺ plus dithiothreitol was not specific for adenylate cyclase, because phosphodiesterase, thiaminediphosphatase and many other proteins were also solubilized. Solubilized adenylate cyclase had a high specific activity and was not activated by either NaF, 5′-guanylyl imidodiphosphate (Gpp[NH]p) or calmodulin. These results suggested that lipid peroxidation of the brain membranes significantly solubilized adenylate cyclase of high specific activity.     

Biophysical consequences of lipid peroxidation in membranes

This article reviews the biophysical consequences of lipid peroxidation in biological membranes. In the lipid domain, lipid peroxidation (a) causes an increase in the order and "viscosity" of the membrane bilayer, particularly at the depth around acyl-carbon 12, (b) changes the thermotropic phase behaviour, (c) decreases the electrical resistance, and (d) facilitates phospholipid exchange between the two monolayers. Upon lipid peroxidation membrane proteins are crosslinked, and their rotational and lateral mobility is decreased. Studies with microsomal cytochrome P-450 suggest protein aggregation but not the increased lipid order to be the major cause of protein immobilization in peroxidized membranes.     

Effect of ionizing radiation and Fe2+-induced peroxidation on the lipid phase of erythrocyte membrane preparations

The fluorescent probes, perilene and diphenyl hexatriene, were used to study changes in the lipid phase of erythrocytic ghosts induced by ionizing radiation (100-1000 Gy) and lipid peroxidation initiated by Fe2+ (5-100 microM). Both of the factors were shown to bring about similar changes in the membrane, that is, an increase in the viscosity of the probe localization sites and a decrease in diphenyl hexatriene fluorescence intensity. During the postirradiation incubation of the exposed membranes they were additionally damaged whereas upon peroxidation, most of the changes occurred after 15-min incubation with Fe2+.     

Methemoglobin-induced lipid peroxidation in model membranes]

Lipid peroxidation induced by methemoglobin in liposomes prepared from lecithin, cardiolipin and their mixtures has been investigated. Using absorption spectroscopy technique it was shown that in the bilayers with low initial oxidation methemoglobin caused the formation of diene conjugates. In the bilayers with high degree of oxidation protein activated cleavage of the available fatty acid hydroperoxides. Hydroperoxides were found to induce the reduction of methemoglobin absorption in the Soret band.     

Effect of Fe2+- and gamma-induced lipid peroxidation on respiration and oxidative phosphorylation in the mitochondria

Lipid peroxidation activated by low concentrations of Fe2+ ions in a medium (1-5 microM) and gamma-quanta (10-50 Gy) stimulates the oxygen consumption and oxidative phosphorylation during the initial period of incubation (10-20 min). With relatively high concentrations of Fe2+ ions and higher radiation doses (50-100 Gy) inhibition of the activity of mitochondria is registered with respect to both indices.     

Carnosine protection of Ca2+ transport against damage induced by lipid peroxidation

The authors studied the protective action of carnosine on sarcoplasmic reticulum (SR) membranes from frog skeletal muscles destroyed by ascorbic acid-dependent lipid peroxidation (LPO). It was demonstrated that addition of carnosine to the incubation medium at a concentration of 25 mM sharply decelerated inactivation of Ca-ATPase of SR membranes, maintaining at the same time the coupling of hydrolysing and transport functions of the Ca-pump. When given at the same concentration carnosine inhibited the accumulation of LPO products reacting with 2-thiobarbituric acid. This effect of carnosine was followed by its utilization.     

Energy-dependent transport of Ca2+ and lipid peroxidation in the sarcoplasmic reticular membranes of rats during hypokinesia

Thirty-day hypokinesia in Wistar rats did not affect the rate of Ca2+ transport and the activity of Ca-ATPase in light and heavy fractions of sarcoplasmic reticulum of primarily white muscles. As hypokinesia was raised up to 90 days, these indicators increased. The intensity of lipid peroxidation in sarcoplasmic reticulum was lower in hypokinetic animals.