Effect of a zinc-deficient diet on mitochondrial and microsomal lipid composition in TEPC-183 plasmacytoma

A zinc-deficient diet caused an increase in microsomal membrane phospholipid levels compared to ad libitum controls. Cholesterol levels were found to be decreased 50% compared to either pair-fed or ad libitum controls, resulting in a sharp decline in the cholesterol/phospholipid ratio. No differences were observed in the distribution of phospholipid classes among all three groups, either in mitochondrial or microsomal membrane fractions. Fatty acid analysis of PC and PE revealed a rise in the 18:2 fraction from zinc-deficient mitochondrial and microsomal membrane fractions. Mitochondrial PE and PC from zinc-deficient animals revealed a rise in the 22:6 fatty acid fraction while microsomal PC also revealed a corresponding decrease in 20:4. None of the zinc-deficient preparations differed significantly from either ad libitum or pair-fed controls in the content of long-chain alk-l-enyl ethers. The results of this study point to an effect of a zinc-deficient diet on lipid metabolism in tumor subcellular membranes which may account for the decreased rate of tumor growth observed in zinc-deficient animals.     

The effect of heavy metals on processes of lipid peroxidation in microsomal membranes from the hepatopancreas of the bivalve mollusc mizuhopecten yessoensis

1. Studies were performed regarding the effects of cadmium and copper on the lipid peroxide contents and on the initial rates of NADPH-dependent and Fe-ascorbic acid-dependent lipid peroxidation in the microsomes of hepatopancreas of the scallop Mizuhopecten yessoensis exposed to these pollutants for 3 weeks.2. The results demonstrated that copper accumulation in hepatopancreas cells was accompanied by a significant increase in hydroperoxide and malondialdehyde contents in microsomal membranes, and by alteration in the both enzymic and non-enzymic lipid peroxidation. Cadmium didn't appear to potentiate lipid peroxidation processes in mollusc tissues.3. Possible reasons for different effects of these metals on lipid peroxidation processes are discussed.     

Mitochondrial lipid peroxidation is influenced by dietary factors in early colon carcinogenesis

Oxidative damage to mitochondrial proteins, lipids, and DNA seem to influence the promotion and progression of tumors. High-fat diets and diets high in iron decrease manganese superoxide dismutase activity, a mitochondrial antioxidant, in colon mucosa. Lipid peroxidation products are low in microsomal preparations from colonic mucosa even under peroxide-inducing conditions. However, damage specific to mitochondrial membranes is unknown. This study was designed to investigate dietary lipid and iron effects on fatty acid incorporation and lipid peroxide formation in mitochondrial membranes of colonic mucosa. Male Fischer rats were fed high-fat diets containing either corn oil or menhaden oil with an iron level of either 35 or 535 mg/kg diet. Animals were given two injections of the colon carcinogen, azoxymethane, or saline. Colon tissue was collected 1 and 6 weeks after injections. Mitochondrial and microsomal fractions were prepared for fatty acid analysis and quantitation of lipid peroxidation products. Results showed that lipid composition of both subcellular fractions were influenced by diet. Fatty acid composition of mitochondria differed from microsomes, but overall saturation remained constant. Peroxidation products in mitochondrial membranes were significantly greater than in microsomal membranes. Dietary treatment significantly affected mitochondrial peroxidation in carcinogen-treated animals. Therefore, mitochondria from colon mucosa are more susceptible to peroxidation than are microsomes, dietary factors influence the degree of peroxidation, and the resulting damage may be important in early colon carcinogenesis.     

Rat lung microsomal lipid peroxidation: effects of vitamin E and reduced glutathione

Lung microsomal membranes that contain the redox active components associated with the mixed-function oxidase system can be peroxidized in vitro. To investigate the characteristics of rat lung microsomal lipid peroxidation, we performed experiments using a variety of peroxidation initiators and microsomes obtained from normal and vitamin E-deficient rats. We found that lung microsomes obtained from normal rats are peroxidized much less than liver microsomes obtained from the same animals. Only initiation systems using very high concentrations of ferrous iron produced any significant peroxidation of normal rat lung microsomes. Lung microsomes obtained from vitamin E-deficient rats were found to be much more susceptible to peroxidation. Glutathione (GSH) was effective in inhibiting peroxidation when lung microsomes from normal rats were peroxidized. GSH was not effective in decreasing peroxidation when microsomes from vitamin E-deficient rats were peroxidized in the same system. We conclude that both GSH and vitamin E protect lung microsomal membranes from peroxidation. Glutathione protection appears to be related to the presence of a sulfhydryl group.     

Lipid peroxidation in liver and colon of methylazoxymethanol treated rats

Weanling male Sprague-Dawley rats were injected via the tail vein with methylazoxymethanol (MAM) acetate at a dose of 70 mg/kg body weight. Measurements of lipid peroxidation were carried out on mitochondrial and microsomal fractions of liver and colonic mucosa at various intervals over the first 24 h following delivery of the carcinogen. Significantly increased levels of peroxidation were observed 3-6 h after treatment in microsomal and mitochondrial fractions of both these tissues. A return to control levels was seen by the end of the first day. These results are discussed in relation to the role of lipid peroxidation in carcinogenesis and the proposed mechanism of tumor prevention by selenium.     

Melatonin reduces rat hepatic macromolecular damage due to oxidative stress caused by delta-aminolevulinic acid

Delta-aminolevulinic acid, precursor of heme, accumulates in a number of organs, especially in the liver, of patients with acute intermittent porphyria. The potential protective effect of melatonin against oxidative damage to nuclear DNA and microsomal and mitochondrial membranes in rat liver, caused by delta-aminolevulinic acid, was examined. Changes in 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels, an index of DNA damage, and alterations in membrane fluidity (the inverse of membrane rigidity) and lipid peroxidation in microsomal and mitochondrial membranes, as indices of damage to lipid and protein molecules in membranes, were estimated. Measurements were made in rat liver after a 2 week treatment with delta-aminolevulinic acid (40 mg/kg b.w., every other day). To test the potential protective effects of melatonin, the indole was injected (i.p. 10 mg/kg b.w.) 3 times daily for 2 weeks. 8-OHdG levels and lipid peroxidation in microsomal membranes increased significantly whereas microsomal and mitochondrial membrane fluidity decreased as a consequence of delta-aminolevulinic acid treatment. Melatonin completely counteracted the effects of delta-aminolevulinic acid. Melatonin was highly effective in protecting against oxidative damage to DNA as well as to microsomal and mitochondrial membranes in rat liver and it may be useful as a cotreatment in patients with acute intermittent porphyria.     

The role of lipid peroxidation in the N-oxidation of 4-chloroaniline.

Irradiation with u.v. light of aerobic aqueous media containing both rabbit liver microsomal fraction and 4-chloroaniline results in N-oxidation of the arylamine. The reaction is severely blocked by exhaustive extraction with organic solvents of the microsomal membranes to remove lipids. Further, scavengers of OH. and O2.-impair the photochemical process. These findings suggest that the observed phenomenon may be closely associated with light-induced lipid peroxidation. Indeed, N-oxidation of 4-chloroaniline is fully preserved when either phospholipid liposomes or dispersed linoleic acid substitute for intact microsomal fraction. Co-oxidation of the amine substrate occurs during iron/ascorbate-promoted lipid peroxidation also, but H2O2 or free OH. radicals do not appear to be involved. Cumene hydroperoxide-sustained rabbit liver microsomal turnover of the amine generates N-oxy product via O2-dependent and -independent pathways; propagation of lipid peroxidation is presumed to govern the former route. Lipid hydroperoxides, either exogenously added to rabbit liver microsomal suspensions or enzymically formed from arachidonic acid in ram seminal-vesicle microsomal preparations, support N-oxidation of 4-chloroaniline. The significance, in arylamine activation, of lipid peroxidation in certain extrahepatic tissues exhibiting but low mono-oxygenase activity is discussed.     

Comparison of cytidinediphospho-sn-1,2-diglyceride transfer from microsomal and liposomal to mitochondrial membranes

Transfer of [3H]CDP-diglycerides from isolated guinea pig liver microsomal and liposomal membranes to guinea pig mitochondrial membranes was studied by incubating microsomal or liposomal membranes carrying [3H]CDP-diglycerides with mitochondrial membranes and determining the CDP-diglyceride-dependent incorporation of sn-3-[14C]glycerolphosphate into mitochondrial [14C]polyglycerophosphatides. A significant difference in the amount of transferred [3H]CDP-diglycerides and the composition of mitochondrial [14C]polyglycerophosphatides was found depending on whether [3H]CDP-diglycerides were transferred from microsomal or liposomal membranes. This amount was around 12% when [3H]CDP-diglycerides were transferred from the microsomal membranes and around 4.6% when they were transferred from the liposomal membranes. Furthermore, about 60% of [14C]phosphatidylglycerol and 35% of [14C]phosphatidylglycerophosphate were found in the microsomes-mitochondria system and about 9% of [14C]phosphatidylglycerol and 79% of [14C]phosphatidylglycerophosphate were found in the liposomes-mitochondria system, establishing an important role for the membrane donor in the transfer of [3H]CDP-diglycerides to mitochondria. Furthermore, if the transfer of [3H]CDP-diglycerides from the microsomal to the mitochondrial membranes was assayed by the determination of [3H]CDP-diglycerides in reisolated mitochondrial membranes without further incorporation into mitochondrial polyglycerophosphates, it amounted to about 38%.     

Superoxide dismutase depletion and lipid peroxidation in rat liver microsomal membranes: correlation with liver carcinogenesis

The depletion of superoxide dismutase in the liver of rats held on a copper-deficient diet for 8 weeks induces two profound modifications in microsomal membrane characteristics. These membranes show: (1) a low degree of peroxidation induced in vitro by both endogenous (NADPH and tert-butylhydroperoxide) and exogenous sources (xanthine/xanthine oxidase) of oxygen radicals as revealed by malondialdehyde and diene-conjugate production; (2) a strong decrease of polyunsaturated and an increase of monounsaturated fatty acid content. These alterations are similar to those found in microsomal membranes from fast-growing hepatomas which exhibit a pronounced saturation of fatty acid pattern and lack superoxide dismutase. These observations support the hypothesis that during hepatocarcinogenesis the loss of superoxide dismutase causes an oxidative stress that increases cellular membrane lipid peroxidation, as a consequence of which the cell responds by synthesizing more saturated fatty acids that permanently modify cell membrane structure and properties.     

Temperature-induced changes of spin-labelled radioactive lipids in isolated guinea pig liver microsomal membranes before and in mitochondrial membranes after their translocation.

Lipids of isolated guinea pig liver microsomal membranes were labelled biosynthetically with isomeric doxyl stearic acid and temperature-induced changes of these membranes were studied by electron spin resonance. A noticeable discontinuity was detected at 10--12 degree C with 12- or 16-doxyl stearic acid containing membrane lipids which was attributed to the spin-labelled lipid--microsomal membrane protein interactions since no such discontinuity was detected in liposomes prepared from total lipid extracts of microsomal membranes. When microsomal membranes containing radioactive isomeric spin-labelled lipids were incubated with unlabelled mitochondria, reisolated mitochondrial membranes contained translocated radioactive isomeric spin-labelled lipids. Temperature-induced changes in these membranes showed no discontinuity with either isomeric doxyl stearic acid derivative, establishing a difference in the environment of translocated lipids in the membrane donor compared with that in the membrane acceptor. Microsomal membranes recovered from translocation experiments showed the same behaviour as the original membranes and exhibited the same discontinuity at 10--12 degree C, establishing that the translocation incubation itself did not alter the spin-labelled lipid interaction within these membranes. Studies of the loss of paramagnetism of spin-labelled lipids in microsomal membranes before and in mitochondrial membranes after their translocation showed a significant difference and suggested that both the outer and the inner mitochondrial membranes might have been involved.     

Lipid peroxidation inhibitory factors in liver and muscle of rat, mouse, and chicken

Glutathione- or sulfhydryl-dependent antioxidant factors that act to prevent lipid peroxidation have been reported in both microsomes and cytoplasm from rat liver. The cytoplasmic factor has been identified in several other tissues and species, but the distribution of the microsomal factor has not been reported. Chicken and mouse livers had much lower activities of the glutathione-dependent membrane-associated and cytoplasmic antioxidant factors than rat liver. Peroxidative damage to membranes has been hypothesized as a mechanism of tissue damage in muscular dystrophy. However, neither the chicken, mouse, nor rat had significant activities of the antioxidant factors in muscle. There was also no significant difference between normal and dystrophic chicken livers in the activity of the antioxidant factors associated with the microsomes or the cytoplasm, nor of the liver microsomal factor in normal and dystrophic mice. The results do not support an important role for the antioxidant factors in the pathogenesis of muscular dystrophy, and raise questions as to whether such factors are physiologically important in species other than rat or in tissues other than liver.     

Complement-component-C3-opsonized immunoglobulin G anti-DNA antibodies do not bind effectively to red blood cells unless aggregated on a high-Mr DNA matrix.

The significance of microsomal vitamin E in protecting against the free-radical process of lipid peroxidation was evaluated with the low-level-chemiluminescence technique in microsomal fractions from vitamin E-deficient and control rats. The induction period that normally precedes the ascorbate/ADP/Fe3+-induced lipid peroxidation was taken as reflecting the microsomal vitamin E content and was found to be 5-6-fold decreased in microsomal fractions from vitamin E-deficient rats. Supplementation of microsomal fractions from vitamin E-deficient rats with exogenous vitamin E partially restores the induction period observed in that from control rats. The decrease in chemiluminescence intensity and the increase in the induction period both correlate linearly with the amount of vitamin E added. However, the efficiency of exogenous vitamin E is about 50-fold lower than that exerted by the naturally occurring vitamin E in microsomal membranes. These observations are discussed in terms of the process of re-incorporation of vitamin E into membranes, the experimental model for lipid peroxidation selected, and the method to evaluate lipid peroxidation, namely low-level chemiluminescence.     

The role of lipid radicals in electron transport and energy transformation

Data given propose two regimes of lipid radicals and oxygen utilization realized in microsomal and mitochondrial membranes. The first one, lipid peroxidation, i.e. interaction of lipid radicals and oxygen is an empty step. In converting this regime to the functional one NADPH-dependent lipid peroxidation is inhibited. A change of this regime to the functional one in microsome demand the presence of hydroxylation substrates. Setting lipid radical-dependent coupling apparatus on phosphorylation in mitochondria occur in the presence of ADP and Pi-phosphorylation substrates.     

Inhibition of phospholipid hydrolysis by phospholipase A2 in microsomal and mitochondrial membranes subjected to lipid peroxidation

The rate of phospholipid hydrolysis in rat liver microsomal and mitochondrial membranes catalyzed by phospholipase A2 was shown to decrease after ascorbate + Fe2+-induced lipid peroxidation. The degree of inhibition was linearly dependent on the amount of lipid peroxidation products (malonyl dialdehyde) accumulated in the membrane. The decreased phospholipid hydrolysis rate in membranes after lipid peroxidation was registered using phospholipases A2 from two sources: porcine pancreas and bee venom. It was established that the inhibitory action of phospholipid peroxidation products was not linked with a direct effect on the enzyme and was not caused by depletion of phospholipase reaction substrates (as a result of lipid peroxidation). A possible role of lateral separation of oxidized and non-oxidized lipid phases in the mechanisms of inhibition of phospholipid hydrolysis by phospholipase A2 is discussed.     

Effects of dietary zinc on free radical generation, lipid peroxidation, and superoxide dismutase in trained mice.

The purpose of this study was to investigate the effects of dietary zinc on free radical generation, lipid peroxidation, and superoxide dismutase (SOD) in exercised mice. In the first part of the study, 48 male weanling mice were randomly divided into three groups. They were fed a zinc-deficient diet containing 1.6 mg/kg zinc or were pair-fed or fed ad libitum a zinc-adequate diet supplemented with 50 mg/kg zinc. Half of each group received an exercise training program that consisted of swimming for 60 min per day in deionized water. The diets and exercise program persisted for 6 weeks. In the second part of the study, 64 mice were fed zinc-deficient diets for 6 weeks, and then one group was fed the zinc-deficient diet for an additional 3 weeks, and the other three groups were fed diets supplemented with 5, 50, and 500 mg/kg zinc, respectively. Half of each group also received the exercise program. Both blood and liver samples were examined. Free radicals in liver were directly detected by electron spin resonance techniques and the extent of lipid peroxidation was indicated by malonic dialdehyde (MDA). Both CuZn-SOD and Mn-SOD were measured. The results showed that exercise training increased the metabolism of zinc, and zinc deficiency induced an increased free radical generation and lipid peroxidation and a decreased hepatic CuZn-SOD activity in exercised mice. Furthermore, although exercise training had no effect on the level of free radicals in zinc-adequate mice, it could increase the hepatic mitochondrial MDA formation further in zinc-deficient animals and zinc deficiency would eliminate the exercise-induced increase in SOD activities which existed in zinc-adequate mice. A total of 50 mg/kg zinc supplemented in the diet was adequate to correct the zinc-deficient status in exercised mice while 5 mg/kg zinc had a satisfactory effect on the recovery of only sedentary zinc-deficient mice. However, 500 mg/kg zinc had a harmful effect on both sedentary and exercised zinc-deficient animals.     

Selective enzymatic radioactive and spin labelling of phospholipids in biological membranes: application to study of temperature-induced changes of microsomal phosphatidylinositols and mitochondrial polyglycerophosphatides.

A new method for the covalent radioactive and spin labelling of lipids within isolated biological membranes has been described in detail and applied to studies of temperature-induced changes of microsomal and mitochondrial membranes. The method is based on the enzymatic use of radioactive substrates carrying covalently bound doxyl derivatives of stearic acid in the biosynthesis of phospholipids in isolated membranes. Radioactive-and spin-labelled lipids bound to the microsomal and mitochondrial membranes were then used as internal probes in monitoring temperature-induced changes of these membranes. Since the analysis of isolated radioactive-and spin-labelled lipids revealed the exact composition of membrane-bound labelled lipids, specific temperature-induced changes were correlated with specific lipids of examined membranes. Phosphatidylinositol of microsomal membranes and polyglycerophosphatides (phosphatidyl-glycerol and cardiolipin) of mitochondrial and inner mitochondrial membranes were found to be involved in the apparent formation of lipid clusters at around 20-30 degrees C. Cardiolipin was found to be involved in the fluidization of inner mitochondrial membranes. These findings are discussed in view of the present state of knowledge of the organization of lipids in biological membranes.     

A comparative study of the interactions of bleomycin with nuclei and purified DNA

Fe(III)-bleomycin associates strongly with rat liver nuclei and binds to nuclear DNA. Metal-free and Cu(II)-bleomycin, however, do not bind to nuclei. The treatment of nuclei with activated iron-bleomycin results in nucleic base and base propenal release from the DNA, and also gives membrane peroxidation. Isolation and quantitation of the base propenals and free bases released subsequent to activated bleomycin treatment reveal an alteration in the stoichiometry of these products compared to those released from purified DNA. With nuclei, significantly less propenal is formed, although the yield of free base is equivalent to that from purified DNA. The membrane peroxidation products from nuclei are the same as those obtained from microsomal membranes treated with activated bleomycin. Superoxide dismutase inhibits the membrane peroxidation but has no effect on the DNA breakage reactions. The results implicate a role for iron in mediating the in vivo action of bleomycin and also reveal a potentially toxic effect, membrane peroxidation, separate from DNA damage.     

Mitochondrial importation of lipids and liponucleotides from microsomes independent of and facilitated by purified cytosol proteins

The mitochondrial importation of microsomal lipids and liponucleotides in the presence and in the absence of partially purified cytosol protein(s) isolated from guinea pig liver was studied by the aid of isomeric (5-, 12-, and 16-(N-oxyl-4',4'-dimethyloxazolidine)stearoyl) spin-labelled radioactive phosphatide acid, phosphatidylcholine, neutral lipids, and CDP-diglycerides. Using a conventional procedure for the protein purification, cytosol protein(s) was purified approximately 1000-fold in respect to its ability to catalyze the translocation of isomeric spin-labelled lipids and liponucleotides from the microsomal to mitochondrial membranes. The highest activity of this protein was exhibited with biosynthesized spin-labelled lipids and liponucleotides bound to the microsomal membranes as substrates and the lowest, with the synthetic liponucleotides and derived lipids bound to the microsomal membranes. The partially purified protein was active in catalyzing the mitochondrial import of phospholipids from microsomes after heat treatment up to 90 degrees C. In addition to the cytosol protein catalyzing mechanism of mitochondrial import of lipids and liponucleotides from microsomal membranes, another cytosol protein independent mechanism of the mitochondrial importation of the same lipids and liponucleotides was also demonstrated in an agreement with our previous reports on the existence of cytosol protein independent intermembranous translocation of phospholipids. These experimental findings are discussed in terms of possible physiological significance and reaction mechanisms involved in the mitochondrial import of lipids and liponucleotides from the microsomal membranes of guinea pig liver.     

Lipid peroxidation increases the molecular order of microsomal membranes

The effect of enzymatic lipid peroxidation on the molecular order of microsomal membranes was evaluated by ESR spectroscopy using the spin probes 5-, 12-, and 16-doxyl-stearic acid. Rat liver microsomal membranes were peroxidized by the NADPH-dependent reaction in the presence of the chelate ADP-Fe3+. Peroxidation resulted in a preferential depletion of polyenoic fatty acids and an increase in the percentage composition of shorter fatty acyl chains. There was no change in the cholesterol/phospholipid ratio of the peroxidized microsomes. The molecular order of both control and peroxidized membranes decreased toward the central region of the bilayer, and the order parameter (S) of each probe was temperature dependent. Peroxidation of the microsomal membrane lipids resulted in an increase in the order parameter determined with the three stearic acid spin probes. Of the three probes, 12-doxylstearic acid was the most sensitive to the changes in membrane organization caused by peroxidation. These data indicate that ESR spectroscopy is a sensitive method of detecting changes in membrane order accompanying peroxidation of membrane lipids.     

Abnormal membrane phospholipid content in subcellular fractions from the Morris 7777 hepatoma.

1. Mitochondrial and microsomal fractions were prepared from normal rat liver and the Morris 7777 hepatoma and characterized by the use of the marker enzymes, succinate dehydrogenase and rotenone-insensitive NADPH-cytochrome c reductase. 2. The phospholipid content per mg membrane protein of Morris 7777 hepatoma mitochondria was increased by 75% as compared with mitochondria from normal rat liver. Microsomes from this poorly-differentiated tumor were found to have a 45% decrease in the content of phospholipid. These abnormalities were independent of tumor size or age. 3. The percent phospholipid content of the subcellular fractions was determined, and revealed an increase in the percent sphingomyelin in both the microsomal and mitochondrial fractions of the tumor. Decreases in the percent phosphatidylcholine and phosphatidylethanolamine were noted in tumor microsomes as compared with normal liver. Diphosphatidylglycerol was not found in significant quantities in the microsomal fraction of this hepatoma line. 4. The content of the various phospholipid classes per mg protein in the respective mitochondrial and microsomal fractions was determined. Large increases in nearly all the major phospholipid classes were found in tumor mitochondria; tumor microsomes were characterized by an increased content of sphingomyelin but the content of nearly all other phospholipids was significantly decreased. These findings suggest the presence of disturbances in the regulation of phospholipid metabolism in subcellular organelle membranes of the Morris 7777 hepatoma.