Genetic polymorphism of the CYP1A1, CYP2E1, GSTM1 and GSTT1 genes and lung cancer susceptibility in a north Indian population

The present study investigates in a experimental system in vitro the relationship between the non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation in rat liver microsomes and nuclei. Chemiluminescence was measured as cpm/mg protein during 180 min at 37 degrees C. Approximately 50-55% of the fatty acids located in rat liver microsomes and nuclei are polyunsaturated with a prevalence of C18:2 n6 and C20:4 n6. The values of total light emission during the non-enzymatic and enzymatic lipid peroxidation were highest in microsomes than in nuclei. A significant decrease of C20:4 n6 and C22:6 n3 in rat liver microsomes and nuclei was observed during the lipid ascorbate-Fe2+-dependent peroxidation, whereas a significant decrease of C20:4 n6 in rat liver microsomes was observed during enzymatic lipid peroxidation. Over the time course studies, analysis of chemiluminescence in microsomes and nuclei demonstrated that the lipid peroxidation in the presence of ascorbate-Fe2+ reach a maximum at 50 and 30 min, respectively, whereas in the presence of NADPH it reachs a maximum at 20 min in both organelles. In liver microsomes and nuclei the peroxidizability index (pi) which indicates the degree of vulnerability to degradation of a selected membrane showed statistically significant differences between control versus ascorbate-Fe2+ when microsomes or nuclei were compared. Our results indicate that non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation are operative in rat liver microsomes and nuclei but the sensitivities of both organelles to lipid peroxidation evidenced by chemiluminescence was greater in the presence of ascorbate-Fe2+ when compared with NADPH.     

Melatonin preserves arachidonic and docosapentaenoic acids during ascorbate-Fe2+ peroxidation of rat testis microsomes and mitochondria

The pineal hormone melatonin (N-acetyl, 5-methoxytryptamine) was recently accepted to act as an antioxidant under both in vivo and in vitro conditions. In this study, we examined the possible preventive effect of melatonin on ascorbate-Fe(2+) lipid peroxidation of rat testis microsomes and mitochondria. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:5 n6. The lipid peroxidation of testis microsomes or mitochondria produced a significant decrease of C20:4 n6 and C22:5 n6. The light emission (chemiluminescence) used as a marker of lipid peroxidation was similar in both kinds of organelles when the control and peroxidized groups were compared. Both long chain polyunsaturated fatty acids were protected when melatonin was incorporated either in microsomes or mitochondria. The melatonin concentration required to inhibit by 100% the lipid peroxidation process was 5.0 and 1.0mM in rat testis microsomes and mitochondria, respectively. IC 50 values calculated from the inhibition curve of melatonin on the chemiluminescence rates were higher in microsomes (4.98 mM) than in mitochondria (0.67 mM). The protective effect observed by melatonin in rat testis mitochondria was higher than that observed in microsomes which could be explained if we consider that the sum of C20:4 n6+C22:5 n6 in testis microsomes is two-fold greater than present in mitochondria.     

Arachidonic acid hydroperoxide stimulates lipid peroxidation in rat liver nuclei and chromatin fractions

Arachidonic acid, the most abundant polyunsaturated fatty acid in rat liver nuclei phospholipids is a major target of free radical attack, which induces lipid peroxidation. The non-enzymatic lipid peroxidation process in intact rat liver nuclei and in several chromatin fractions indicated that the most sensitive fatty acid for peroxidation is arachidonic acid C20:4 n-6. In this study, the effect of different amounts of arachidonic acid hydroperoxide on the lipid peroxidation of rat liver nuclei and chromatin fractions was studied; rat liver nuclei and chromatin fractions deprived of exogenous added hydroperoxide were utilized as control. The addition of arachidonic acid hydroperoxide to liver nuclei produces a marked increase in light emission that was hydroperoxide concentration dependent. The maximal peak of chemiluminescence displayed by the different chromatin fractions analyzed was observed between 20 and 80 min of incubation. The highest value of light emission was displayed by the high-density chromatin fractions, the 27.5 K fraction showed intermediate values of light emission, whereas the lowest density fraction produced very low chemiluminescence. A high correlation between arachidonic acid hydroperoxide concentration and chemiluminescence in the different chromatin fractions was observed. AC is Members of Carrera del Investigador Científico, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.     

Comparison of the ability of ferric complexes to catalyze microsomal chemiluminescence, lipid peroxidation, and hydroxyl radical generation

The interaction of microsomes with iron and NADPH to generate active oxygen radicals was determined by assaying for low level chemiluminescence. The ability of several ferric complexes to catalyze light emission was compared to their effect on microsomal lipid peroxidation or hydroxyl radical generation. In the absence of added iron, microsomal light emission was very low; chemiluminescence could be enhanced by several cycles of freeze-thawing of the microsomes. The addition of ferric ammonium sulfate, ferric-citrate, or ferric-ADP produced an increase in chemiluminescence, whereas ferric-EDTA or -diethylenetriaminepentaacetic acid (detapac) were inhibitory. The same response to these ferric complexes was found when assaying for malondialdehyde as an index of microsomal lipid peroxidation. In contrast, hydroxyl radical generation, assessed as oxidation of chemical scavengers, was significantly enhanced in the presence of ferric-EDTA and -detapac and only weakly elevated by the other ferric complexes. Ferric-desferrioxamine was essentially inert in catalyzing any of these reactions. Chemiluminescence and lipid peroxidation were not affected by superoxide dismutase, catalase, or competitive hydroxyl radical scavengers whereas hydroxyl radical production was decreased by the latter two but not by superoxide dismutase. Chemiluminescence was decreased by the antioxidants propylgallate or glutathione and by inhibiting NADPH-cytochrome P-450 reductase with copper, but was not inhibited by metyrapone or carbon monoxide. The similar pattern exhibited by ferric complexes on microsomal light emission and lipid peroxidation, and the same response of both processes to radical scavenging agents, suggests a close association between chemiluminescence and lipid peroxidation, whereas both processes can be readily dissociated from free hydroxyl radical generation by microsomes.     

Comparative studies on lipid peroxidation of microsomes and mitochondria obtained from different rat tissues: effect of retinyl palmitate

The effect of retinyl palmitate on the polyunsaturated fatty-acid composition, chemiluminescence and peroxidizability index of microsomes and mitochondria obtained from rat liver, kidney, brain, lung and heart, was studied. After incubation of microsomes and mitochondria in an ascorbate Fe++ system (120 min at 37 degrees C) it was observed that the total cpm/mg protein originated from light emission: chemiluminescence was lower in liver microsomes, mitochondria and kidney microsomes in the vitamin A group than in the control group. In mitochondria obtained from control rats, the most sensitive fatty acids for peroxidation were arachidonic acid C20:4 n6 in liver and docosahexaenoic acid C22:6 n3 in kidney and brain. In microsomes obtained from control rats, the most sensitive fatty acids for peroxidation were linoleic acid C18:2 n6 and C20:4 n6 in liver and C22:6 n3 in kidney. Changes in the most polyunsaturated fatty acids were not observed in organelles obtained from lung and heart. As a consequence the peroxidizability index, a parameter based on the maximal rate of oxidation of fatty acids, showed significant changes in liver, kidney and brain mitochondria, while in microsomes changes were significant in liver and kidney. These changes were less pronounced in membranes derived from rats receiving vitamin A. Our results confirm and extend previous observations that indicated that vitamin A may act as an antioxidant protecting membranes from deleterious effects.     

Antioxidant activity of the pyridoindole stobadine in liposomal and microsomal lipid peroxidation.

Stobadine, a pyridoindole derivative, is an efficient inhibitor of lipid peroxidation in phosphatidylcholine liposomes and in rat liver microsomes treated with iron/ADP/NADPH as pro-oxidant. Accumulation of thiobarbituric acid-reactive substances (TBARS) or low-level chemiluminescence were taken as a measure of lipid peroxidation and 5 microM stobadine doubled the duration of the lag phase preceding the onset of rapidly increasing chemiluminescence. Inhibition of lipid peroxidation was not observed with tocopherol-deficient microsomes, suggesting that the antioxidant effect of stobadine depends on vitamin E in the membrane. The cis(-) isomer was most effective, with the cis(+) and trans(rac) as well as dehydro- or acetyl derivatives being less active. In liposomes, the presence of reductant (NADPH or ascorbate) protects from the loss of stobadine.     

A high involvement of O2- possibly generated in inner membranes for iron-induced microsomal lipid peroxidation.

The lipid peroxidation of and the O2- generation by rat liver microsomes in the presence of NADPH or both NADPH and Fe3+ were determined by thiobarbituric acid-reacting substance formation and by chemiluminescence intensities with a cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3, 7-dihydroimidazo[1,2-a]pyrazin-3-one(MCLA), as a chemiluminescence probe. Judging from the experiments with various inhibitors on the O2- generation and the lipid peroxidation, O2- generated, at intramembranous site, by cytochrome P-450 system is considered to be highly involved in the iron-induced lipid peroxidation.     

Pulmonary surfactant protein A inhibits the lipid peroxidation stimulated by linoleic acid hydroperoxide of rat lung mitochondria and microsomes

Reactive oxygen species play an important role in several acute lung injuries. The lung tissue contains polyunsaturated fatty acids (PUFAs) that are substrates of lipid peroxidation that may lead to loss of the functional integrity of the cell membranes. In this study, we compare the in vitro protective effect of pulmonary surfactant protein A (SP-A), purified from porcine surfactant, against ascorbate-Fe(2+) lipid peroxidation stimulated by linoleic acid hydroperoxide (LHP) of the mitochondria and microsomes isolated from rat lung; deprived organelles of ascorbate and LHP were utilized as control. The process was measured simultaneously by chemiluminescence as well as by PUFA degradation of the total lipids isolated from these organelles. The addition of LHP to rat lung mitochondria or microsomes produces a marked increase in light emission; the highest value of activation was produced in microsomes (total chemiluminescence: 20.015+/-1.735 x 10(5) cpm). The inhibition of lipid peroxidation (decrease of chemiluminescence) was observed with the addition of increasing amounts (2.5 to 5.0 microg) of SP-A in rat lung mitochondria and 2.5 to 7.5 microg of SP-A in rat lung microsomes. The inhibitory effect reaches the highest values in the mitochondria, thus, 5.0 microg of SP-A produces a 100% inhibition in this membranes whereas 7.5 microg of SP-A produces a 51.25+/-3.48% inhibition in microsomes. The major difference in the fatty acid composition of total lipids isolated from native and peroxidized membranes was found in the arachidonic acid content; this decreased from 9.68+/-1.60% in the native group to 5.72+/-1.64% in peroxidized mitochondria and from 7.39+/-1.14% to 3.21+/-0.77% in microsomes. These changes were less pronounced in SP-A treated membranes; as an example, in the presence of 5.0 microg of SP-A, we observed a total protection of 20:4 n-6 (9.41+/-3.29%) in mitochondria, whereas 7.5 microg of SP-A produced a 65% protection in microsomes (5.95+/-0.73%). Under these experimental conditions, SP-A produces a smaller inhibitory effect in microsomes than in mitochondria. Additional studies of lipid peroxidation of rat lung mitochondria or microsomes using equal amounts of albumin and even higher compared to SPA were carried out. Our results indicate that under our experimental conditions, BSA was unable to inhibit lipid peroxidation stimulated by linoleic acid hydroperoxide of rat lung mitochondria or microsomes, thus indicating that this effect is specific to SP-A.     

Inhibition of protein carbonyl formation and lipid peroxidation by glutathione in rat liver microsomes.

The peroxidation of rat liver microsomal lipids is stimulated in the presence of iron by the addition of NADPH or ascorbate and is inhibited by the addition of glutathione (GSH). The fate of GSH and the oxidative modification of proteins under these conditions have not been well studied. Rat liver microsomes were incubated at 37 degrees C under 95% O2:5% CO2 in the presence of 10 microM ferric chloride, 400 microM ADP, and either 450 microM ascorbic acid or 400 microM NADPH. Lipid peroxidation was assessed in the presence 0, 0.2, 0.5, 1, or 5 mM GSH by measuring thiobarbituric acid reactive substance (TBARS) and oxidative modification of proteins by measuring protein thiol and carbonyl groups. GSH inhibited TBARS and protein carbonyl group formation in both ascorbate and NADPH systems in a dose-dependent manner. Heat denaturing of microsomes or treatment with trypsin resulted in the loss of this protection. The formation of protein carbonyl groups could be duplicated by incubating microsomes with 4-hydroxynonenal. Ascorbate-dependent peroxidation caused a loss of protein thiol groups which was diminished by GSH only in fresh microsomes. Both boiling and trypsin treatment significantly decreased the basal protein thiol content of microsomes and enhanced ascorbate-stimulated lipid peroxidation. Protection against protein carbonyl group formation by GSH correlated with the inhibition of lipid peroxidation and appeared not to be due to the formation of the GSH conjugate of 4-hydroxynonenal as only trace amounts of this conjugate were detected. Ninety percent of the GSH lost after 60 min of peroxidation was recoverable as borohydride reducible material in the supernatant fraction. The remaining 10% could be accounted for as GSH-bound protein mixed disulfides. However, only 75% of the GSH lost during peroxidation appeared as glutathione disulfide, suggesting that some was converted to other soluble borohydride reducible forms. These data support a role for protein thiol groups in the GSH-mediated protection of microsomes against lipid peroxidation.     

The relationship between chemiluminescence and lipid peroxidation in rat hepatic microsomes.

Studies were carried out to determine the relationship between NADPH- and ascorbate-initiated chemiluminescence (CL) and lipid peroxidation (LP) in rat hepatic microsomes. NADPH-initiated CL and LP become maximal 15 min after addition of NADPH to the microsomes and ascorbate-initiated CL and LP become maximal 90 to 120 min following addition of ascorbate. There are four lines of evidence to indicate that both NADPH- and ascorbate-initiated chemiluminescence are related to lipid peroxidation. (i) The time courses for the increases in CL and in LP are identical. (ii) There is a linear relationship between total (integral) or maximal CL and LP. (iii) Drug substrates which inhibit LP also inhibit CL in a quantitatively similar manner. (iv) Inhibitors of lipid peroxidation, such as Co²⁺, Mn²⁺, Hg²⁺, para-chloromercuribenzenesulfonic acid, and EDTA, also inhibit chemiluminescence. The results of these experiments indicate that chemiluminescence initiated in hepatic microsomes by either NADPH or ascorbate is directly proportional to lipid peroxidation.     

Effect of chronic ethanol treatment on the t-butyl hydroperoxide-dependent lipid peroxidation in rat liver

1. The effect of chronic ethanol consumption on the level of the t-butyl hydroperoxide (Bu'OOH)-induced lipid peroxidation in rat liver homogenate and subcellular fractions was measured using chemiluminescence technique and malondialdehyde formation. 2. It was shown that under the action of ethanol the rate of lipid peroxidation was decreased in the whole and "postnuclear" liver homogenates. 3. Ethanol significantly decreased the intensity of lipid peroxidation in microsomes, but did not affect the Bu'OOH-dependent process in mitochondria. 4. The level of lipid peroxidation was reduced after incubation of the total particulate fraction (mitochondria plus microsomes) with the undialysed cytosol from ethanol-treated rat liver. Dialysis of the cytosol prevented depressive effect of ethanol treatment on lipid peroxidation. 5. Reduced glutathione (0.1-1.0 mM) was shown to decrease the rate of lipid peroxidation in rat liver microsomes, but did not affect its level in mitochondria. 6. Pyrazole injections to rats reduced and phenobarbital treatment increased the level of the Bu'OOH-dependent lipid peroxidation in liver microsomes. 7. The data obtained indicate that the Bu'OOH-dependent lipid peroxidation is not an appropriate marker of the ethanol-induced oxidative stress in rat liver cells.     

Lipid peroxidation dependent aldrin epoxidation in liver microsomes, hepatocytes and granulation tissue cells

Lipid peroxidation activity was determined in liver microsomes, hepatocytes and cultured granuloma cells by measuring ethane and pentane production with an improved capillary gas chromatographic method. Lipid peroxidation initiated by ferrous ions and NADPH produced significantly more hydrocarbons at 4% O2 than under atmospheric (21% O2), hyperoxic or hypoxic conditions. In liver microsomes ferrous ions and ascorbic acid stimulated the non-enzymatic lipid peroxidation and concomitantly the epoxidation of aldrin. The results demonstrate that epoxidation of aldrin can be triggered by the iron initiated lipid peroxidation.     

NADPH-dependent drug redox cycling and lipid peroxidation in microsomes from human term placenta

1. NADPH-dependent iron and drug redox cycling, as well as lipid peroxidation process were investigated in microsomes isolated from human term placenta. 2. Paraquat and menadione were found to undergo redox cycling, catalyzed by NADPH:cytochrome P-450 reductase in placental microsomes. 3. The drug redox cycling was able to initiate microsomal lipid peroxidation in the presence of micromolar concentrations of iron and ethylenediaminetetraacetate (EDTA). 4. Superoxide was essential for the microsomal lipid peroxidation in the presence of iron and EDTA. 5. Drastic peroxidative conditions involving superoxide and prolonged incubation in the presence of iron were found to destroy flavin nucleotides, inhibit NADPH:cytochrome P-450 reductase and inhibit propagation step of lipid peroxidation. 6. Reactive oxo-complex formed between iron and superoxide is proposed as an ultimate species for the initiation of lipid peroxidation in microsomes from human term placenta as well as for the destruction of flavin nucleotides and inhibition of NADPH:cytochrome P-450 reductase as well as for impairment of promotion of lipid peroxidation under drastic peroxidative conditions.     

Selective inhibition of the non-enzymatic lipid peroxidation of phosphatidylserine in rod outer segments by α-tocopherol

In the present study it was investigated if a-tocopherol shows protection against in vitro lipid peroxidation of phospholipids located in rod outer segment membranes (ROS). After incubation of ROS in an ascorbate-Fe²⁺ system, at 37°C during 160 min, the total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of -tocopherol. The fatty acid composition of total lipids isolated from rod outer segment membranes was substantially modified when subjected to non-enzymatic lipid peroxidation with a considerable decrease of docosahexaenoic acid (22:6 n-3). The incorporation of -tocopherol (0.35 mol/mg protein) produce a 43.37% inhibition of the lipid peroxidation process evaluated as chemiluminiscence (total cpm originated in 160 min). The phospholipid species containing the highest amount of docosahexaenoic acid: phosphatidyletanolamine and phosphatidylserine were more affected than phosphatidylcholine during the lipid peroxidation process. Not all phospholipids, however, were equally protected after the addition of -tocopherol to the incubation medium. Phosphatidylcholine and phosphatidyletanolamine, were not protected by -tocopherol, the vitamin provides selective antioxidant protection only for phosphatidylserine. These results indicate that -tocopherol may act as antioxidant protecting rod outer segment membranes from deleterious effect by a selective mechanism that diminishes the loss of docosahexaenoic acid from phosphatidylserine.     

Relative efficacies of α-tocopherol, N-acetyl-serotonin, and melatonin in reducing non-enzymatic lipid peroxidation of rat testicular microsomes and mitochondria

In this study, we examined the relative efficacies of alpha-tocopherol, N-acetyl-serotonin, and melatonin in reducing ascorbate-Fe(2+) lipid peroxidation (LPO) of rat testicular microsomes and mitochondria. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids (PUFAs) C20:4 n6 and C22:5 n6. The LPO of testicular microsomes or mitochondria produced a significant decrease of C20:4 n6 and C22:5 n6. Both long-chain PUFAs were protected when the antioxidants were incorporated either in microsomes or mitochondria. By comparison of the IC50 values obtained between alpha-tocopherol and both indolamines, it was observed that alpha-tocopherol was the most efficient antioxidant against the LPO induced by ascorbate-Fe(2+) under experimental conditions in vitro, IC50 values from the inhibition of alpha-tocopherol on the chemiluminescence were higher in microsomes (0.14 mM) than in mitochondria (0.08 mM). The protective effect observed by alpha-tocopherol in rat testis mitochondria was higher compared with microsomes, associated with the higher amount of [C20:4 n6] + [C22:5 n6] in microsomes than that in mitochondria. Melatonin and N-acetyl-serotonin were more effective in inhibiting the LPO in mitochondria than that in microsomes. Thus, a concentration of 1 mM of both indolamines was sufficient to inhibit in approximately 70% of the light emission in mitochondria, whereas a greater dosage of 10 times (10 mM) was necessary to produce the same effect in microsomes. It is proposed that the vulnerability to LPO of rat testicular microsomes and mitochondria in the presence of both indolamines is different because of the different proportion of PUFAs in these organelles.     

Chemiluminescence associated with the oxidative metabolism of salicylic acid in rat liver microsomes

Rat liver microsomal suspension (1 mg protein per ml) was incubated at 37 degrees C with 5 mM salicylic acid and 0.2 mM NADPH. The amounts of thiobarbituric acid reactive substances (TBARS) and 2,5-dihydroxybenzoic acid (2,5-DHB), an oxidative metabolite of salicylic acid increased with the incubation time. Simultaneously spontaneous chemiluminescence (CL) was found to be generated there. The addition of SKF-525A, an inhibitor of cytochrome P450 (P450), to the reaction mixture inhibited the CL generation together with the inhibition of the oxidative metabolism. The anti-oxidants and singlet oxygen scavengers like N,N-diphenylphenylenediamine (DPPD) and histidine suppressed the CL generation. The addition of 1,4-diazabicyclo [2.2.2] octane (DABCO), a singlet oxygen quencher, to the reaction mixture generating CL enhanced CL transiently and then CL decreased markedly. Thus CL observed here may possibly originate from the singlet oxygen. The CL generation was suggested to be closely related with salicylic acid-induced lipid peroxidation, and to be coupled with the oxidative metabolism mediated by P450 in rat liver microsomes.     

Effect of the microsomal system on interconversions between hydroquinone, benzoquinone, oxygen activation, and lipid peroxidation

Our previous results indicated that cytochrome P450 destruction by benzene metabolites was caused mainly by benzoquinone (Soucek et al., Biochem. Pharmacol. 47 (1994) 2233-2242). The aim of this study was to investigate the interconversions between hydroquinone, semiquinone, and benzoquinone with regard to both spontaneous and enzymatic processes in order to test the above hypothesis. We have also studied the participation of hydroquinone and benzoquinone in OH radicals formation and lipid peroxidation as well as the role of ascorbate and transition metals. In buffered aqueous solution, hydroquinone was slowly oxidized to benzoquinone via a semiquinone radical. This conversion was slowed down by the addition of NADPH and completely stopped by microsomes in the presence of NADPH. Benzoquinone was reduced to semiquinone radical at a significantly higher rate and this conversion was stimulated by NADPH and more effectively by microsomes plus NADPH while semiquinone radical was quenched there. In microsomes with NADPH. both hydroquinone and benzoquinone stimulated the formation of OH radicals but inhibited peroxidation of lipids. Ascorbate at 0.5-5 mM concentration also produced significant generation of OH radicals in microsomes. Neither hydroquinone nor benzoquinone did change this ascorbate effect. On the contrary, 0.1-1.0 mM ascorbate stimulated peroxidation of lipids in microsomes whereas presence of hydroquinone or benzoquinone completely inhibited this deleterious effect of ascorbate. Iron-Fe2+ apparently played an important role in lipid peroxidation as shown by EDTA inhibition, but it did not influence OH radical production. In contrast, Fe3+ did not influence lipid peroxidation, but stimulated OH radical production. Thus, our results indicate that iron influenced the above processes depending on its oxidation state, but it did not influence hydroquinone/benzoquinone redox processes including the formation of semiquinone. It can be concluded that interconversions between hydroquinone and benzoquinone are influenced by NADPH and more effectively by the complete microsomal system. Ascorbate, well-known antioxidant produces OH radicals and peroxidation of lipids. On the other hand, both hydroquinone and benzoquinone appear to be very efficient inhibitors of lipid peroxidation.     

Effect of seminal plasma antioxidant on lipid peroxidation in spermatozoa, mitochondria and microsomes

Seminal plasma antioxidant inhibited ascorbate/iron-induced lipid peroxidation in spermatozoa, brain and liver mitochondria. The concentration required to produce inhibition in brain and liver mitochondria was high. Denaturation of spermatozoa resulted in complete loss of antioxidant action. Maintenance of native structure was essential for action of seminal plasma antioxidant in spermatozoal lipid peroxidation. The antioxidant inhibited NADPH, Fe3+-ADP induced lipid peroxidation in microsomes and consequences of lipid peroxidation such as glucose-6-phosphatase inactivation were prevented by presence of antioxidant. It did not inhibit microsomal lipid peroxidation induced by ascorbate and iron and xanthine-xanthine oxidase.     

Effects of beta-carotene and alpha-tocopherol on radical-initiated peroxidation of microsomes.

Rat liver microsomal membranes were exposed to either beta-nicotinamide adenine dinucleotide phosphate (NADPH), adenosine 5'-diphosphate (ADP), and Fe+3 or to azocompounds, and the antioxidant activities of beta-carotene and alpha-tocopherol were studied. Lipid peroxidation was monitored either by malondialdehyde (MDA) formation in the thiobarbituric acid assay at 535 nm or by hydroperoxide formation at 234 nm, after high-pressure liquid chromatography (HPLC) separation of phospholipid hydroperoxides. The radical initiators, water-soluble 2,2'-azobis(2-amidinopropane) (AAPH) and lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile (AMVN), when thermally decomposed at 37 degrees C under air, produced a constant rate of lipid peroxidation in microsomes and lag times inversely related to their concentrations. Using 25 mM AAPH, beta-carotene suppressed lipid peroxidation at a concentration of 50 nmol/mg protein; using 24 mM AMVN, an inhibition of MDA formation was observed at a concentration of only 5 nmol/mg protein. Inhibition by beta-carotene did not produce a clearly defined lag phase. During AAPH-induced lipid peroxidation, beta-carotene was consumed linearly, and high levels of the antioxidant were still present at the end of 45 min of incubation. Using NADPH/ADP/Fe+3, protection by beta-carotene was observed at 10 nmol/mg protein. alpha-Tocopherol effectively suppressed both MDA and hydroperoxide formation in a dose-dependent manner when either NADPH/ADP/Fe+3 or azocompounds were used. These effects were observed at very low concentrations of the added alpha-tocopherol, ranging from 2 to 3 nmol/mg protein. When the lag times were measurable (AAPH and AMVN), they were directly proportional to the concentration of alpha-tocopherol and revealed the presence of endogenous antioxidants in the microsomal membranes. Different temporal relationships between the loss of alpha-tocopherol and lipid peroxidation were observed in relation to the prooxidant used. A substantial depletion of about 70% of endogenous alpha-tocopherol preceded the propagation phase when induced by the azocompounds, while only 20% of antioxidant disappeared at the beginning of the peroxidation when induced by NADPH/ADP/Fe+3. Although our results show that both beta-carotene and alpha-tocopherol suppress the peroxidation of microsomal membranes, their antioxidant efficacy is influenced by several factors, including the type of radical initiator involved and the site and rate of radical production.     

Lipid peroxidation in rat liver microsomes. II. Response of hydroperoxide formation to iron concentration

When rat liver microsomes were incubated with NADPH, the major products were hydroperoxides which increased with time indicating that endogenous iron content is able to promote lipid peroxidation. The addition of either 5 microM Fe2+ or Fe3+ ions strongly enhanced the hydroperoxide formation rate. However, due to the hydroperoxide breakdown, hydroperoxide concentration decreased with time in this case. Higher ferrous or ferric iron concentration did not change the situation much, in that both hydroperoxide breakdown and formation were similar to those when NADPH only was present in the incubation medium. After lipid peroxidation, analysis of fatty acids indicated that the highest amount of peroxidized PUFA occurred in the presence of 5 microM of either Fe2+ or Fe3+. This analysis also showed that after 8 min incubation with low iron concentration, PUFA depletion was about 77% of that observed after 20 min, whereas without any iron addition or in the presence of 30 microM of either Fe3+, PUFA decrease was only about 37% of that observed after 20 min. As far as the optimum Fe2+/Fe3+ ratio required to promote the initiation of microsomal lipid peroxidation in rat liver is concerned, the highest hydroperoxide formation was observed with a ratio ranging from 0.5 to 2. These results indicate that microsomal lipid peroxidation induced by endogenous iron is speeded up by the addition of low concentrations of either Fe2+ or Fe3+ ions, probably because free radicals generated by hydroperoxide breakdown catalyze the propagation process. In experimental conditions unfavourable to hydroperoxide breakdown the principal process is that of the initiation of lipid peroxidation.