Changes in peroxidation under conditions of 3,4-benzypyrene-induced carcinogenesis

The activities of enzymatic systems generating and destroying peroxides and the lipid peroxide content in neoplastic rat liver and 3,4-benzpyrene-induced sarcoma were studied. The tumour was characterized by high activity of glutathione peroxidase and low activity of catalase. No urate- and glycolate oxidases or ascorbat dependent peroxidation of lipids and lipid peroxides were found in the tumour. In the liver of neoplastic animals the activities of glutathione peroxidase and NADPH-dependent system of microsomal phospholipid peroxidation and the lipd peroxides content were increased, whereas the activities of catalase and urate oxidase were decreased.     

Effect of ultraviolet rays on peroxidation and its regulation in the rat liver

The indirect effect of rat skin ultraviolet (UV) irradiation on lipid peroxidation and enzymatic systems of the liver has been studied. The processes of lipid peroxidation have been intensified after 72 hours of UV-irradiation, which is evidently due both to the activation of enzymatic system of initiation and propagation of lipid peroxidation and to the parallel decrease of the activity of enzymatic system regulation of given process in liver.     

Glutathione and lipid peroxidation in the aging rat

1. Tissue extracts were prepared from liver, kidney, heart, brain, lung and spleen of male Sprague-Dawley rats of different ages (1-36 months); each of the extracts was analyzed for reduced glutathione (GSH) and lipid peroxides. 2. At all ages the GSH content in the liver was 3-10 times higher than that in other tissues. 3. In the old (36 months) rat the GSH content of all the tissues studied were lower (35-60%) than that in 2.5 month old rat. 4. The lipid peroxides levels increased by age in all tissues studied. 5. These findings indicate that general characteristics of aging tissue may include a decrease in GSH content and increase in lipid peroxides showing a decrease in reducing potential in senescence.     

Lipoprotein Oxidation and Induction of Ferroxidase Activity in Stored Human Extracellular Fluids

It was observed that during the storage of human extracellular fluids at – 20°C the azide-inhibitable ferroxidase activity of caeruloplasmin declined, whilst a new azide-resistant ferroxidase activity (ARFA) developed. The literature suggested that storage-induced ARFA might be due to either a poorly defined enzymatic activity of a low density lipoprotein (LDL) or to lipid peroxides formed within the different lipoprotein fractions. To study this further, the major lipoprotein classes were separated from human serum by density gradient centrifugation. After storage of the lipoprotein fractions, it was found that the LDL fraction had the highest specific activity of ARFA and the highest content of lipid peroxidation products, as assessed by diene conjugates. The ARFA of LDL correlated with its content of diene conjugates and TBA reactive material, which initially suggested that the Fe(II) oxidising activity of peroxidised LDL arose from the reduction of peroxides by Fe(II) in the classical reaction between the metal ion and free radical reduction of lipid peroxides. However. steady state kinetic analysis indicated an enzymic role of LDL in Fe(II) oxidation, with lipid peroxides acting as a substrate for the enzyme. These results indicate that LDL may contain a peroxidase activity. catalysing the oxidation of Fe(II) by lipid peroxides, as well as a ferrous oxidase activity where O₂ is the oxidising substrate.     

Studies on lipid peroxidation in normal and tumour tissues. The Novikoff rat liver tumour.

A study has been made of the factors that contribute to the decreased rates of lipid peroxidation under different pro-oxidant conditions in intact Novikoff tumour cells, and in microsomal suspensions prepared from Novikoff tumour cells, compared with isolated normal rat hepatocytes and microsomal suspensions prepared from normal rat liver. The pro-oxidant conditions were the addition of either NADPH, NADPH + ADP + iron, NADPH + CCl4 or ascorbate+iron to the experimental systems used, or exposure to gamma-radiation. Contributory factors to the lower rates of lipid peroxidation observed include: a significant decrease in the polyunsaturated fatty acid content of Novikoff cells or Novikoff microsomes; the decreases are especially marked for the C20:4 and C22:6 fatty acids; a very marked reduction in NADPH-cytochrome c reductase; and no detectable content of cytochrome P-450. Another, and in our opinion critical, contribution to the diminished rate of lipid peroxidation in the tumour material is the substantial increase in alpha-tocopherol relative both to total lipid and to methylene-interrupted double bonds in fatty acids. Moreover, the alpha-tocopherol is the major contributor to lipid-soluble chain-breaking antioxidant in lipid extracts of normal liver and of Novikoff tumour material.     

Paracetamol-stimulated lipid peroxidation in isolated rat and mouse hepatocytes

Treatment of isolated hepatocytes from 3-methylcholanthrene induced rats with 1 mM paracetamol has been found to greatly decrease cellular reduced glutathione (GSH) content and to promote lipid peroxidation, evaluated as malonaldehyde (MDA) production and conjugated diene absorbance. A similar dosing of hepatocytes from phenobarbital-induced or normal rats is ineffective in that respect. On the other hand, the aspecific stimulation of the cytochrome P-450-mediated paracetamol activation due to acetone addition further increases GSH depletion as well as MDA production.Isolated hepatocytes with basal low GSH content are also more susceptible to paracetamol-induced lipid peroxidation, indicating that the rate of the drug metabolism and the cellular GSH content are critical factors in the determination of such peroxidative attack.In isolated mouse liver cells paracetamol does not require preliminary cytochrome P-450 induction to stimulate MDA formation, even at concentrations ineffective in rat cells.However, 5 mM paracetamol, despite a great depletion of cellular GSH content, does not promote MDA formation either in the rat or in the mouse hepatocytes. This effect may be due to the ability of paracetamol to scavenge lipid peroxides under defined conditions, as tested in various lipid peroxidizing systems.Membrane leakage of lactate dehydrogenase (LDH) is evident in paracetamol treated cells undergoing lipid peroxidation, but not when MDA formation is inhibited by high doses of the drug or by addition of antioxidants such as α-tocopherol and diphenylphenylenediamine (DPPD).Nevertheless in these conditions the covalent binding of activated paracetamol metabolites is not affected, suggesting that lipid peroxidation might play a role in the pathogenesis of liver damage following paracetamol overdose.     

Antioxidative and metabolic responses to extended cold exposure in rats

In this work, we investigated whether extended cold exposure increases oxidative damage and susceptibility to oxidants of rat liver, heart, kidney and lung which are metabolically active tissues. Moreover in this study the effect of cold stress on some of the lipid metabolic mediators were studied in rat experimental model. Male albino Sprague-Dawley rats were randomly divided into two groups: The control group (n=12) and the cold-stress group (n=12). Tissue superoxide dismutase (SOD), catalase (CAT), glutathion S-transferase (GST) and glutathion reductase (GR) activities and glutathion (GSH) were measured using standard protocols. The biochemical analyses for total lipid, cholesterol, trigliceride, HDL, VLDL and LDL were done on autoanalyzer. In cold-stress groups SOD activity was decreased in the lung whereas it increased in the heart and kidney. CAT activity was significantly decreased (except liver) in all the tissues in treated rats. GST activity of cold-induced rats increased in liver and heart while decreased in the lung. GR activity was significantly decreased (except in liver) in all the tissues in cold-stressed rats. GSH level was significantly increased in the heart but decreased in the lung of animals exposed to cold when compared to controls. It was found that among the groups trigliceride, total lipid, HDL and VLDL parameters varied significantly but cholesterol and LDL had no significant variance. In this study, we found that exposure of extended (48 h) cold (8 degrees C) caused changes both in the antioxidant defense system (as tissue and enzyme specific) and serum lipoprotein profiles in rats.     

Direct detection of radical generation in rat liver nuclei on treatment with tumour-promoting hydroperoxides and related compounds

EPR spin trapping has been employed to directly detect radical production in isolated rat nuclei on exposure to a variety of hydroperoxides and related compounds which are known, or suspect, tumour promoters. The hydroperoxides, in the absence of reducing equivalents, undergo oxidative cleavage, generating peroxyl radicals. In the presence of NADPH (and to a lesser extent NADH) reductive cleavage of the OO bond generates alkoxyl radicals. These radicals undergo subsequent rearrangements and reactions (dependent on the structure of the alkoxyl radical), generating carbon-centred radicals. Acyl peroxides and peracids appear to undergo only reductive cleavage of the OO bond. With peracids this cleavage can generate aryl carboxyl (RCO₂·) or hydroxyl radicals (HO·); with acyl peroxides, aryl carboxyl radicals are formed and, in the case of t-butyl peroxybenzoate, alkoxyl radicals (RO·). The radicals detected with each peroxide are similar in type to those detected in the rat liver microsomal fraction, although the extent of radical production is lower. The subsequent reactions of the initially generated radicals are similar to those determined in homogenous chemical systems, suggesting that they are in free solution. Experiments with NADPH/NADH, heat denaturation of the nuclei and various inhibitors suggest that radical generation is an enzymatic process catalysed by haemproteins, in particular cytochrome P-450, and that NADPH/cytochrome P-450 reductase is involved in the reductive cleavage of the OO bond. The generation of these radicals by the rat liver nuclear fraction is potentially highly damaging for the cell due to the proximity of the generating source to DNA. Several previous studies have shown that some of the radicals detected in this study, such as aryl carboxyl and aryl radicals, can damage DNA, via various reactions which results in the generation of strand breaks and adducts to DNA bases: these processes are suggested to play an important role in the tumour promoting activity of these hydroperoxides and related compounds.     

Enhanced heme oxygenase activity increases the antioxidant defense capacity of guinea pig liver upon acute cobalt chloride loading: comparison with rat liver

Changes in the activity of so-called oxidative stress defensive enzymes, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and heme oxygenase, as well as changes in lipid peroxidation and reduced glutathione levels, were measured in guinea pig and rat liver after acute cobalt loading. Cobalt chloride administration produced a much higher degree of lipid peroxidation in guinea pig than in rat liver compared with the control animals. The intrahepatic reduced glutathione content in control guinea pig was higher than that in rat, but was equally decreased in both species after cobalt administration. The enzymatic scavengers of free radicals, superoxide dismutase, catalase and glutathione peroxidase, were significantly decreased in rat liver after acute cobalt loading, and as a compensatory reaction, the heme oxygenase activity was increased (seven-fold). In guinea pig liver, only superoxide dismutase activity was depleted in response to cobalt-induced oxidative stress, while catalase and glutathione peroxidase were highly activated and the heme oxygenase activity was dramatically increased (13-fold). It is assumed that enhanced heme oxygenase activity may have important antioxidant significance by increasing the liver oxidative-stress defense capacity.     

Regulation of transmethylation by an S-adenosylmethionine binding protein

A protein which has a high affinity for S-adenosylmethionine (SAM) has been partially purified from rat liver. This binding protein stimulates both the rate and extent of product formation when added to both a lipid methylating system, phosphatidylethanolamine: SAM-N-methyltransferase, and an RNA methylating system, the t-RNA methylase complex from rat liver. The S-adenosylmethionine binding protein by itself has no enzymatic activity in either transmethylation system.     

Some mechanisms of antihypoxic action of taurine]

It is shown that preliminary taurine treatment prevents the disturbances of energy metabolism in the brain, heart and liver tissues of Wistar rats with acute hypoxic hypoxia. Administration of taurine restored to normal the parameters of adenine pool: the concentration of ATP increased within the cytoplasm, while that of ADP and AMP diminished; mitochondrial respiration proceeded more rapidly; the concentrations of pyruvate and malate decreased; isocitrate dehydrogenase activity, P/O and NAD/NADH ratios increased. Taurine treatment resulted in a decreased level of lipid peroxides in the rat tissues with hypoxia. The role of intracellular calcium content and biomembranes structure changes as the mechanisms of taurine action on energy metabolism and lipid peroxidation is discussed.     

Oxidative damage following cerebral ischemia depends on reperfusion - a biochemical study in rat

The extent of brain injury during reperfusion appears to depend on the experimental pattern of ischemia/reperfusion. The goals of this study were: first, to identify the rate of free radicals generation and the antioxidant activity during ischemia and reperfusion by means of biochemical measurement of lipid peroxidation (LPO) and both enzymatic (superoxid dismutase - SOD, catalase - CAT, glutathion peroxidase - GPx) and non-enzymatic antioxidants activity (glutathione - GSH); and second, to try to find out how the pattern of reperfusion may influence the balance between free radical production and clearance. Wistar male rats were subject of four-vessel occlusion model (Pulsinelly & Brierley) cerebral blood flow being controlled by means of two atraumatic arterial microclamps placed on carotid arteries. The level of free radicals and the antioxidant activity were measured in ischemic rat brain tissue homogenate using spectrophotometrical techniques. All groups subjected to ischemia shown an increase of LPO and a reduction of the activity of enzymatic antioxidative systems (CAT, GPx, SOD) and non-enzymatic systems (GSH). For both groups subjected to ischemia and reperfusion, results shown an important increase of LPO but less significant than the levels found in the group with ischemia only. Statistically relevant differences (p<0.01) between continuous reperfusion and fragmented reperfusion were observed concerning the LPO, CAT, SOD and GSH levels, oxidative aggresion during fragmented reperfusion being more important.     

Follow-up study of lipid peroxides, superoxide dismutase and glutathione peroxidase in the synovial membrane, serum and liver of young and old mice with collagen-induced arthritis

Because reactive oxygen species (ROS) are generally believed to play an important role in tissue injury in rheumatoid arthritis, we examined the levels of lipid peroxides, superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in the synovial membrane, serum and liver of young (8 wk) and old (12 mo) mice with collagen-induced arthritis. In the synovial membrane, serum and liver, lipid peroxide levels of both young and old mice were increased beginning on the 3rd day after the onset of arthritis. SOD activity, which scavenges O2- and inhibits lipid peroxidation, rose markedly in the synovial membrane of young mice in parallel with the increase in lipid peroxide levels, but not so markedly in old mice. Liver GSH-Px activity, which metabolizes already formed lipid peroxides, also rose in young arthritic mice to a greater degree than in old mice. This study suggests that in inflammatory synovial lesions, lipid peroxides are generated due to an increase in ROS concentration, with resultant cytotoxicity, and that younger animals or humans can prevent this unfavorable reaction more effectively than aged ones by enzyme induction. The hypothesis that lipid peroxides formed in the oxidative lesions of the primary organ are released into the serum, trapped by the liver and metabolized there is further supported by the present study.     

The tricarboxylate carrier from rat liver mitochondria. Purification, reconstitution and kinetic characterization

The tricarboxylate carrier from rat liver mitochondria has been purified and reconstituted into phospholipid vesicles. Its activity has been characterized by both a radioactive citrate uptake assay and a coupled enzymatic assay. A Km of 40 microM and a Vmax of 1.56 mumol x min-1 x mg-1 have been determined for the carrier. Cholesterol levels of between 5-10% of total lipid content are shown to cause a decrease in carrier activity.     

Cytochrome oxidase induction after oxidative stress induced by adriamycin in liver of rats fed with dietary olive oil.

The influence of different kinds of dietary fat (8%) and of endogenous lipid peroxidation with regard to cytochrome c oxidase activity and cytochrome a + a3 concentrations in mitochondria from rat liver has been investigated. It was possible to confirm that the dietary fat induced higher phospholipid degradation in mitochondrial membranes; moreover an endogenous oxidative stress induced by adriamycin was able to increase the peroxidative effects. We have found that the peroxidative effects could sometimes induce an apparent enhancement of cytochrome oxidase activity due to a significant increase of cytochrome a + a3 content. This finding lets us suppose that both changes in the lipid environment and some peroxidation damage could occur in the membrane as a consequence of the fat assumed. Furthermore we should suggest that an induction of the synthesis of cytochrome a + a3 might be related to an enhanced production of peroxides at membrane level.     

Growth-related lipid peroxidation in tumour microsomal membranes and mitochondria.

Microsomes and mitochondria isolated from Morris hepatomas 3924A (fast-growing) and 44 (slow-growing) and Ehrlich ascites tumour cells exhibit a NADPH-dependent peroxidation of endogenous lipids lower than that of the corresponding fractions from rat liver. Moreover, the O2- and ascorbate-dependent lipid peroxidations are decreased in microsomes from the two Morris hepatomas. The peroxidative activity appears to be inversely related to the growth rate of the tumours. It is suggested that the low susceptibility of tumour membranes to peroxidative agents may be a factor responsible for the high mitotic activity of this tissue.     

Inhibition of lipid oxidation by serum and albumin

The influence of serum and albumin on enzymatic and non-enzymatic lipid oxidation was investigated. Intensity of oxidation was measured as the amount of oxygen consumed by the sample and by quantitation of malonaldehyde formed during breakdown of lipid peroxides. Non-enzymatic lipid oxidation was stimulated by ascorbic acid or ferrous ions and enzymatic by NADPH-dependent oxidase, 15-lipoxygenase and 12-lipoxygenase. Albumin inhibits lipid oxidation only when pure fatty acid (arachidonic or linoleic) is the substrate for this oxidation. Serum was a stronger inhibitor than an equivalent amount of albumin and it also inhibited oxidation of a mixture of lipids from liver microsomes. It is concluded that serum contains two antioxidant factors: albumin which binds fatty acids and probably another factor which is a true antioxidant.     

Changes in lipid peroxide levels and activity of reactive oxygen scavenging enzymes in skin, serum and liver following UVB irradiation in mice.

The effect of acute UVB on the generation of reactive oxygen species (ROS) in the skin and the induction of ROS scavenging enzymes in situ was examined. Lipid peroxide levels and the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) and D-glucose-6-phosphate dehydrogenase (G-6-P-D) were determined in the skin, serum, and liver of ICR mice subjected to 1400 mJ/cm2 of acute UVB irradiation. In irradiated skin, lipid peroxides were increased at 3 and 24 hr after irradiation, whereas the four ROS scavenging enzymes were generally decreased during the first 48 hr after irradiation. In the serum, lipid peroxides showed an increase at 3 hr, but enzyme activities remained negligible. In the liver, lipid peroxides showed similar behaviour to that in skin. GSH-Px activity in the liver was decreased during the first 24 hr, whereas G-6-P-D showed substantial fluctuation and SOD and catalase activities showed no change. These data are consistent with a model in which lipid peroxides generated in the UVB-irradiated lesions are transported to the liver and there metabolized by the scavenging enzymes induced in situ.     

Lipid metabolism and structure-activity features of the endoplasmic reticulum membrane in regenerating rat liver]

The relationship between the neutral lipid and phospholipid metabolism and some structure-function peculiarities of regenerating rat liver endoplasmic reticulum membranes (13 hours after surgery, i.e., corresponding to the G1-period of the cell cycle) was studied. There was an increase in the degree of the endoplasmic reticulum membrane development and the nonesterified fatty acid (NFA) and triglyceride (TG) content in regenerating rat liver microsomes. The relative specific radioactivity of neutral lipid and phospholipid fractions in regenerating rat liver microsomes was lower than in control animals, presumably due to the high rate of the microsomal lipid exchange in the regenerating liver with other cell organelles. The changes in the lipid content and rate of their metabolism in the regenerating rat liver were associated with the increase in the membrane microviscosity and the decrease in the activity of the membrane-bound enzyme (glucose-6-phosphatase). The differences in the time-dependent changes in the synthesis and metabolism of lipids in the NFA and TG fractions may be regarded as an endogenous factor determining the structure-function peculiarities of endoplasmic reticulum membranes.     

Reduction of various lipid hydroperoxides by rat liver homogenate

Rates of disappearance of hydroperoxy groups of various lipid peroxides added to rat liver homogenate differed from each other. The hydroperoxy group of linoleic acid disappeared rapidly, while those of L-3-phosphatidyl choline dilinoleoyl and trilinolenin disappeared slowly. The hydroperoxy groups of cholesterol linoleate were stable in the homogenate. Most of the conjugated dienes of these lipid peroxides remained. The hydroperoxy groups of the unsaturated fatty acids of the phosphatidyl choline were found to be changed to hydroxy groups as analyzed by high-performance liquid chromatography.