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.     

Polychlorinated biphenyls-induced lipid peroxidation as measured by thiobarbituric acid-reactive substances in liver subcellular fractions of rats

Rats were given a 0.05% polychlorinated biphenyls (PCB) diet supplemented with adequate nutrients for 10 days and not only PCB-induced lipid peroxidation as measured by thiobarbituric acid (TBA)-reactive substances but also variations of lipid peroxides scavengers in liver and its subcellular fractions (nuclei and cell debris, mitochondrial, microsomal and cytosolic fractions) were investigated. The lipid peroxidation in liver and subcellular fractions in the PCB-treated group increased significantly except in the nuclei and cell debris fraction. The increase in lipid peroxidation in the microsomal fraction appeared to be associated in part with the decrease in vitamin E (alpha-tocopherol) content and induction of drug-metabolizing enzymes. In the cytosolic fraction, the total lipid content increased, glutathione peroxidase (GSHPx) activity decreased and the quantity of free radical-reactive substances suppressing lipid peroxidation was low as measured by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) value. From these results, the increase in lipid peroxidation in the cytosolic fraction in the PCB-treated group was ascribed to the abundance and availability of oxidizable substrate attended with fatty liver, to the decline in GSHPx activity, and to the insufficiency in antioxygenic activity as observed by the decrease in the DPPH value.     

Effects of diffusible products of peroxidation of rat liver microsomal lipids

The effects on cellular structures of products of peroxidation of rat liver microsomal lipids were investigated. A system containing actively peroxidizing liver microsomal fraction was separated from a revealing or target system by a dialysis membrane. The target system, contained in the dialysis tube, consisted of either intact cells (erythrocytes) or subcellular fractions (liver microsomal fraction). When liver microsomal fractions were incubated with NADPH (or an NADPH-generating system), lipid peroxidation, as measured by the amount of malonaldehyde formed, occurred very rapidly. The malon-aldehyde concentration tended to equilibrate across the dialysis membrane. When the target system consisted of erythrocytes, haemolysis occurred abruptly after a lag phase. The lysis was greatly accelerated when erythrocytes from vitamin E-deficient rats were used, but no haemolysis was observed when erythrocytes from vitamin E-treated rats were used. When, in the same system, freshly prepared liver microsomal fractions were exposed to diffusible factors produced by lipid peroxidation, the glucose 6-phosphatase activity markedly decreased. A similar decrease in glucose 6-phosphatase activity, as well as a smaller but significant decrease in cytochrome P-450, was observed when the target microsomal fractions were exposed to diffusible factors derived from the peroxidation of liver microsomal lipids in a separate preincubation step. These and additional experiments indicated that the toxicological activity is relatively stable. Experiments in which the hepatic microsomal fractions destined for lipid peroxidation contained radioactively labelled arachidonic acid, previously incorporated into the membranes, showed that part of the radioactivity released from the microsomal fraction into the incubation medium entered the dialysis tube and was recovered bound to the constituents of the microsomal fractions of the target system. These results indicate that during the course of the peroxidation of liver microsomal lipids toxic products are formed that are able to induce pathological effects at distant loci.     

Age peculiarities in changes of free radical oxidation processes in the brain of rats with hypothyroidism

The objective of the present experiment was to study age peculiarities of free radical protein oxidation and lipid peroxidation in brain of 1.5-month-old and 12-month-old rats with drug-induced hypothyroidism. It has been shown that hypothyroidism in both 1.5-month and 12-month old rat is accompanied by the oxidative stress in the brain. It manifests by an increase of content of lipid peroxidation products and protein carbonyls in mitochondrial and microsomal fractions. Hypothyroidism decreases the prooxidant effect of exercises on the brain mitochondria.     

Extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex. III. ACTH-induced temporal subcellular redistributions of steroid precursors to corticosterone

Cholesterol, pregnenolone, progesterone, 11-deoxycorticosterone (11-DOC) and corticosterone were quantitated in subcellular fractions isolated from in vivo adrenocorticotropin (ACTH)-stimulated rat adrenal zona fasciculata/reticularis. Six adrenal subcellular fractions separated by discontinuous sucrose gradient centrifugation (lipid, 0.125 M sucrose, cytosolic, microsomal, mitochondrial and nuclear) were extracted with alkaline ether/ethanol and assayed by high pressure liquid chromatography (HPLC). Lipid fractions contained the major cholesterol stores, while most pregnenolone and progesterone was found in lipid, microsomal and mitochondrial fractions. The 0.125 M sucrose and cytosol fractions together contained approximately 75% of the total 11-DOC and corticosterone. The five steroids were only present in small amounts in organelle fractions containing steroidogenic enzymes. Homogenate and lipid fraction cholesterol decreased between 10 and 15 min and again 30 min after ACTH injection. In the homogenate, lipid, microsomal and mitochondrial fractions, pregnenolone and progesterone were increased after ACTH injection; peak pregnenolone and progesterone concentrations were often measured in adrenal gland sucrose, cytosolic, microsomal and mitochondrial fractions 15 to 20 min after rats were injected with ACTH. Although ACTH increased 11-DOC and corticosterone in all but the mitochondrial and nuclear fractions, the sucrose, cytosolic and microsomal 11-DOC, and cytosolic corticosterone increased most dramatically. In many fractions, peak 11-DOC and corticosterone concentrations were most often observed between the 10 and 15 min periods and again at 30 min.     

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.     

A STUDY OF THE EFFECTS OF STATIC AND EXTREMELY LOW FREQUENCY MAGNETIC FIELDS ON LIPID PEROXIDATION PRODUCTS IN SUBCELLULAR FIBROBLAST FRACTIONS

Cultured fibroblasts isolated from murine livers by tissue trypsinization were exposed to a static magnetic field (0.490 T) and to extremely low frequency (ELF) magnetic field (50 Hz, 0.020 T). The cultures were exposed to magnetic fields on four consecutive days for exposure times of 2, 4, 8, 16, 32, and 64 min. After such exposures and obtaining of fibroblast subcellular fractions, lipid peroxidation product—malondialdehyde (MDA) was measured. Increased peroxidation of fibroblasts' membrane structures exposed to an ELF magnetic field was observed in subcellular fractions—microsomal, mitochondrial, and nuclear. No changes in peroxidation of membrane structures were found in fibroblasts exposed to a static magnetic field.     

Age- and sex-related differences in lipid peroxidation of mouse cardiac and skeletal muscles

1. The purpose of the present study was to characterize age- and sex-related changes in lipid peroxidation capacities and enzymatic antioxidants of cardiac and skeletal muscles in NMRI-mice (Mus musculus). 2. Lipid peroxidation rates (unstimulated and enzymatic/iron-stimulated) strongly decreased in skeletal muscle during ageing. 3. Unstimulated lipid peroxidation rate but not that of stimulated, also decreased in cardiac muscle. 4. The total level of Fe2+/ascorbate-stimulated non-enzymatic lipid peroxidation was not, however, affected by ageing. 5. The activity of catalase slightly increased in cardiac muscle and that of glutathione peroxidase in skeletal muscle during ageing. 6. Unstimulated lipid peroxidation rate was significantly higher in the skeletal muscle of male than female mice. 7. Correspondingly, the Fe2+/ascorbate-stimulated lipid peroxidation capacities of microsomal and mitochondrial fractions of skeletal muscle were significantly higher in male mice. 8. The activity of glutathione peroxidase as well as the concentration of lipofuscin were higher in the cardiac muscles of female than male mice.     

Effect of a zinc-deficient diet on lipid peroxidation in liver and tumor subcellular membranes

The effect of a zinc-deficient diet on lipid peroxidation in liver and tumor mitochondrial and microsomal membrane preparations from BALB/c mice was investigated. Mitochondrial and microsomal membranes from both tissues displayed increased rates of in vitro peroxidation, both enzymatic and nonenzymatic. Measurement of in vivo peroxidation, using diene conjugation as an index of measurement revealed slight increases in tissues from zinc-deficient animals that were not statistically significant. Serum lipoperoxides analyzed from all three groups revealed no significant differences. The results point to an alteration in the peroxidation potential of mitochondrial and microsomal membranes due to zinc deficiency which may be related to an alteration in fatty acid composition.     

Loss of latent activity of liver microsomal membrane enzymes evoked by lipid peroxidation. Studies of nucleoside diphosphatase, glucose-6-phosphatase, and UDP glucuronyltransferase

The effects of lipid peroxidation on latent microsomal enzyme activities were examined in NADPH-reduced microsomes from phenobarbital-pretreated male rats. Lipid peroxidation, stimulated by iron or carbon tetrachloride, was assayed as malondialdehyde formation. Independent of the stimulating agent of lipid peroxidation, latency of microsomal nucleoside diphosphatase activity remained unaffected up to microsomal peroxidation equivalent to the formation of about 12 nmol malondialdehyde/mg microsomal protein. However, above this threshold a close correlation was found between lipid peroxidation and loss of latent enzyme activity. The loss of latency evoked by lipid peroxidation was comparable to the loss of latency attainable by disrupting the microsomal membrane by detergent. Loss of latent enzyme activity produced by lipid peroxidation was also observed for microsomal glucose-6-phosphatase and UDPglucuronyltransferase. In contrast to nucleoside diphosphatase, however, both enzymes were inactivated by lipid peroxidation, as indicated by pronounced decreases of their activities in detergent-treated microsomes. According to the respective optimal oxygen partial pressure (po2) for lipid peroxidation, the iron-mediated effects on enzyme activities were maximal at a po2 of 80 mmHg and the one mediated by carbon tetrachloride at a po2 of 5 mmHg. Under anaerobic conditions no alterations of enzyme activities were detected. These results demonstrate that loss of microsomal latency only occurs when peroxidation of the microsomal membrane has reached a certain extent, and that beyond this threshold lipid peroxidation leads to severe disintegration of the microsomal membrane resulting in a loss of its selective permeability, a damage which should be of pathological consequences for the liver cell. Because of its resistance against lipid peroxidation nucleoside diphosphatase is a well-suited intrinsic microsomal parameter to estimate this effect of lipid peroxidation on the microsomal membrane.     

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.     

Cholate solubilization of liver microsomal membrane components which promote NADPH-supported lipid peroxidation.

NADPH-supported lipid peroxidation monitored by malondialdehyde (MDA) production in the presence of ferric pyrophosphate in liver microsomes was inactivated by heat treatment or by trypsin and the activity was not restored by the addition of purified NADPH-cytochrome P450 reductase (FPT). The activity was differentially solubilized by sodium cholate from microsomes, and the fraction solubilized between 0.4 and 1.2% sodium cholate was applied to a Sephadex G-150 column and subfractionated into three pools, A, B, and C. MDA production was reconstituted by the addition of microsomal lipids and FPT to specific fractions from the column, in the presence of ferric pyrophosphate and NADPH. Pool B, after removal of endogenous FPT, was highly active in catalyzing MDA production and the disappearance of arachidonate and docosahexaenoate, and this activity was abolished by heat treatment and trypsin digestion, but not by carbon monoxide. The rate of NADPH-supported lipid peroxidation in the reconstituted system containing fractions pooled from Sephadex G-150 columns was not related to the content of cytochrome P450. p-Bromophenylacylbromide, a phospholipase A2 inhibitor, inhibited NADPH-supported lipid peroxidation in both liver microsomes and the reconstituted system, but did not block the peroxidation of microsomal lipid promoted by iron-ascorbate or ABAP systems. Another phospholipase A2 inhibitor, mepacrine, poorly inhibited both microsomal and pool-B'-promoted lipid peroxidation, but did block both iron-ascorbate-driven and ABAP-promoted lipid peroxidation. The phospholipase A2 inhibitor chlorpromazine, which can serve as a free radical quencher, blocked lipid peroxidation in all systems. The data presented are consistent with the existence of a heat-labile protein-containing factor in liver microsomes which promotes lipid peroxidation and is not FPT, cytochrome P450, or phospholipase A2.     

Kinetics of NADPH-induced lipid peroxidation in rat liver microsomal fractions as a function of age

The kinetics of NADPH-induced lipid peroxidation in hepatic rough and smooth microsomes have been studied in rats ranging in age from 1 day to 2 years. Apparent Km and Vmax for NADPH and the extent of lipid peroxidation show that lipid peroxidation potential is low at birth, increases during postnatal development, and decreases during senescence. Our results indicate that this trend may be due to changes in phospholipid content and NADPH cytochrome c reductase activity in microsomal fractions.     

The effect of orally administered secondary autoxidation products of linoleic acid on the activity of detoxifying enzymes in the rat liver

Radioactive secondary autoxidation products of linoleic acid were administered orally to rats and the incorporation of radioactive substances into lipids was investigated in the liver. The radioactive substances were significantly incorporated into hepatic mitochondrial and microsomal lipids 12 h after the administration. 80% of the radioactivity in mitochondria was detected in neutral lipids. The radioactivity in microsomal neutral lipids significantly decreased and the activity in phospholipids increased 12 h after the administration. On the other hand, contents of lipid peroxide and thiobarbituric acid reactive substances in liver were significantly increased by 40% at 15 h after the administration of the secondary autoxidation products. Activity of marker enzymes used for an indication of the hepatic injury was also elevated. Glutathione peroxidase activity increased 3-fold and catalase activity increased 1.5-fold. Activity of mitochondrial NAD-dependent aldehyde dehydrogenase, however, was decreased by 50%. It seems likely that the secondary autoxidation products orally administered are detoxified in the hepatic mitochondria, metabolized to neutral lipids, and further metabolized to phospholipids in microsomes, while as the incorporated secondary autoxidation products induces hepatic injury by lipid peroxidation.     

Inhibition of microsomal lipid peroxidation by glutathione and glutathione transferases B and AA. Role of endogenous phospholipase A2.

Lipid peroxidation in vitro in rat liver microsomes (microsomal fractions) initiated by ADP-Fe3+ and NADPH was inhibited by the rat liver soluble supernatant fraction. When this fraction was subjected to frontal-elution chromatography, most, if not all, of its inhibitory activity could be accounted for by the combined effects of two fractions, one containing Se-dependent glutathione (GSH) peroxidase activity and the other the GSH transferases. In the latter fraction, GSH transferases B and AA, but not GSH transferases A and C, possessed inhibitory activity. GSH transferase B replaced the soluble supernatant fraction as an effective inhibitor of lipid peroxidation in vitro. If the microsomes were pretreated with the phospholipase A2 inhibitor p-bromophenacyl bromide, neither the soluble supernatant fraction nor GSH transferase B inhibited lipid peroxidation in vitro. Similarly, if all microsomal enzymes were heat-inactivated and lipid peroxidation was initiated with FeCl3/sodium ascorbate neither the soluble supernatant fraction nor GSH transferase B caused inhibition, but in both cases inhibition could be restored by the addition of porcine pancreatic phospholipase A2 to the incubation. It is concluded that the inhibition of microsomal lipid peroxidation in vitro requires the consecutive action of phospholipase A2, which releases fatty acyl hydroperoxides from peroxidized phospholipids, and GSH peroxidases, which reduce them. The GSH peroxidases involved are the Se-dependent GSH peroxidase and the Se-independent GSH peroxidases GSH transferases B and AA.     

Studies on the hyperplasia (''regeneration'') of the rat liver following partial hepatectomy. Changes in lipid peroxidation and general biochemical aspects.

Using the experimental model of partial hepatectomy in the rat, we have examined the relationship between cell division and lipid peroxidation activity. In rats entrained to a regime of 12 h light/12 h dark and with a fixed 8 h feeding period in the dark phase, partial hepatectomy is followed by a rapid regeneration of liver mass with cycles of synchronized cell division at 24 h intervals. The latter phenomenon is indicated in this study by pulses of thymidine kinase activity having maxima at 24 h, 48 h and 72 h after partial hepatectomy. Microsomes prepared from regenerating livers show changes in lipid peroxidation activity (induced by NADPH/ADP/iron or by ascorbate/iron), which is significantly decreased relative to that in microsomes from sham-operated controls, again at 24 h, 48 h and 72 h after the operation. This phenomenon has been investigated with regard to possible underlying changes in the content of microsomal fatty acids, the microsomal enzymes NADPH:cytochrome c reductase and cytochrome P-450, and the physiological microsomal antioxidant alpha-tocopherol. The cycles of decreased lipid peroxidation activity are apparently due, at least in part, to changes in microsomal alpha-tocopherol content that are closely associated in time with thymidine kinase activity.     

NADPH-dependent lipid peroxidation and its effects on aminopyrine N-demethylation in subcellular fractions of human neonatal liver.

NADPH-dependent lipid peroxidation was determined in humans, using subcellular fractions of livers obtained from newborn infants. As reported for other species, activity was concentrated in the microsomal fraction and was similar to that found in the rat. High activity of lipid peroxidation induced by iron decreased aminopyrine N-demethylation and slightly reduced linearity time for the reaction. Compared with the rat, however, human microsomes were more resistant to the effects of lipid peroxidation. If liped peroxidation occurs in vivo it is unlikely to affect drug oxidation to any great degree in human infants.     

Inhibition by coenzyme Q of ethanol- and carbon tetrachloride-stimulated lipid peroxidation in vivo and catalyzed by microsomal and mitochondrial systems

The ability of coenzyme Q to inhibit lipid peroxidation in intact animals as well as in mitochondrial, submitochondrial, and microsomal systems has been tested. Rats fed coenzyme Q prior to being treated with carbon tetrachloride or while being treated with ethanol excrete less thiobarbituric acid-reacting material in the urine than such rats not fed coenzyme Q. Liver homogenates, mitochondria, and microsomes isolated from rats treated with carbon tetrachloride and ethanol catalyze lipid peroxidation at rates which exceed those from animals also fed coenzyme Q. The rate of lipid peroxidation catalyzed by submitochondrial particles isolated from hearts of young, old, and endurance trained elderly rats was inversely proportional to the coenzyme Q content of the submitochondrial preparation in assays in which succinate was employed to reduce the endogenous coenzyme Q. Reduced, but not oxidized, coenzyme Q inhibited lipid peroxidation catalyzed by rat liver microsomal preparations. These results provide additional evidence in support of an antioxidant role for coenzyme Q.     

Changes in thermal phase transition of various membranes during temperature acclimation in Tetrahymena

Changes in the thermal phase transition temperature of membrane lipids were studied by X-ray wide-angle diffraction during adaptation of Tetrahymena pyriformis to a lower growth temperature. After a shift in growth temperature from 39 to 15 degrees C, the phase transition temperature was lowered gradually in microsomal and pellicular phospholipids, whereas that in mitochondrial phospholipids was unchanged for 10 h after the temperature shift. Only a small decrease in the transition temperature of mitochondrial phospholipids was observed, even after 24 h following the shift. Transition temperatures of microsomal, pellicular and mitochondrial phospholipids reached the growth temperature (15 degrees C) about 6, 10 and 24 h after the temperature shift. The temperature dependence of the solid phase in membrane phospholipids was estimated from the 4.2 A peak of the X-ray diffraction pattern. In the case of the phospholipids extracted from cells grown at 39 degrees C, the solid phase was increased upon lowering temperature in a similar manner in all three membrane fractions: mitochondria, pellicles and microsomes. However, in the case of the phospholipids from cells exposed to a lower growth temperature (15 degrees C) for 10 h, the increase in the solid phase was significantly smaller in mitochondrial phospholipids than in two other membrane fractions. The difference in the thermal behaviour of mitochondrial lipid from pellicular and microsomal lipids is discussed in terms of phase transition and phase separation.     

Lipid peroxidation in the liver of carcinogen-resistant rats

Recently, we developed a new strain of rats that exhibit marked resistance to the hepatotoxic and carcinogenic actions of 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) and some other carcinogens. In this work, we compared lipid peroxidation in the liver of these carcinogen-resistant (R) rats and the parental Donryu strain rats that are sensitive (S) to hazardous actions of these carcinogens. The liver microsomal fractions of the R group contained less amounts of polyunsaturated fatty acids. Microsomal lipid peroxidation in the presence of exogenous NADPH was much lower in R rats than in S rats. Liver microsomes of R rats were much less active than those of S rats also in producing 4-hydroxynonenal, carbonyl compounds and conjugated diene. The hepatic contents of ascorbic acid, glutathione, alpha-tocopherol and coenzyme Q in the R rats were similar to those in S rats. The activities of the free radical scavenger enzymes, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), in the two groups were also similar. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are both thought to function in disposal of these cytotoxic aldehydes. The liver microsomal and mitochondrial ALDH activities of the two groups were similar. The ADH activity of the liver cytosolic fraction of R rats was nearly twice that of S rats, as measured with 4-hydroxynonenal as substrate. The higher ADH activity may explain the decreased lipid peroxidation in R rats at least partly, if this enzyme is involved in lipid peroxidation.