A possible mechanism of naproxen-induced lipid peroxidation in rat liver microsomes

Previous papers from our laboratory report that naproxen and salicylic acid induced lipid peroxidation in rat liver microsomes, however, the mechanism is still unclear. In the present paper, ferrous iron release, nicotinamide-adenine dinucleotide phosphate reduced form (NADPH) oxidation and hydrogen peroxide (H2O2) formation have been measured to find out which mechanisms are involved in naproxen- and salicylic acid-induced lipid peroxidation. While the increase of ferrous iron release was observed with high concentrations of naproxen, salicylic acid did not stimulate ferrous iron release. Neither of these drugs stimulated NADPH oxidation and H2O2 formation. However hexobarbital and perfluorohexane, known as uncouplers of cytochrome P450, stimulated microsomal NADPH oxidation, O2 consumption, H2O2 formation and water (H2O) formation involving four-electron oxidase reaction. These results suggest that ferrous iron release contributes to naproxen-induced microsomal lipid peroxidation and that naproxen and salicylic acid are not uncouplers of cytochrome P450. Apparently H2O2 does not play an important role in naproxen- and salicylic acid-induced microsomal lipid peroxidation.     

Carbon tetrachloride-induced lipid peroxidation of rat liver microsomes in vitro.

Characteristics of carbon tetrachloride-induced lipid peroxidation of rat liver microsomes and effect on microsomal enzymes were studies in vitro. Microsomes isolated from well-perfused livers and washed with EDTA-containing medium exhibited low endogenous lipid peroxidation when incubated in a phosphate buffer (> 0.1 M) in the presence of NADPH, whereas carbon tetrachloride stimulated to a great extent the peroxidation under these conditions. The stimulation was dependent on the concentration of NADPH, neither NADH nor ascorbic acid being replaced. The stimulatory action by bromotrichloromethane was more marked than that by carbon tetrachloride, however chloroform had no stimulatory action. N,N-Diphenyl-p-phenylene diamine, diethyldithiocarbamate and disulfiram inhibited carbon tetrachloride-induced lipid peroxidation in low concentrations. Inhibitions by thiol compounds and EDTA were weaker. Ferricyanide, cytochrome c and vitamine K₃ inhibited the stimulation by carbon tetrachloride while no inhibition was seen with carbon monoxide. An increase in the degree of carbon tetrachloride-induced lipid peroxidation resulted in a coincidental decrease in microsomal cytochrome P-450 content accompanying a parallel loss in aminopyrine demethylase activity, while NADH-ferricyanide dehydrogenase and NAD(P)H-eytochrome c reductase activities, and cytochrome b₅ content remained unaffected. Similar results were obtained when microsomes were peroxidized with NADPH in combination with ferric chloride and pyrophosphate. Regarding the mechanism of hepatotoxic action of carbon tetrachloride, these results support the hypothesis of lipid peroxidation.     

Mechanism of paraquat-stimulated lipid peroxidation in mouse brain and pulmonary microsomes.

Paraquat-stimulated NADPH-dependent lipid peroxidation in mouse brain and pulmonary microsomes was inhibited by superoxide dismutase and singlet oxygen quenchers, but not by catalase or hydroxyl radical scavengers. MnCl2, which might form a salt with unsaturated lipid, inhibited the lipid peroxidation in brain microsomes, but not that in pulmonary microsomes. These findings suggest that activated oxygen species, especially superoxide and singlet oxygen, may play a major role in the stimulation of microsomal lipid peroxidation by paraquat in both brain and lung, and that the nature of the lipids exposed to peroxidative attack may be different in microsomes of the two organs.     

Potentiation by carbon tetrachloride of NADPH-dependent lipid peroxidation in lung microsomes.

NADPH-dependent lipid peroxidation occurs in rat lung microsomes in vitro. Expressed per wet weight of tissue we found that lung had only $\text{1}\text{100}$ the activity of liver. However, examination of the rate of malonyl dialdehyde production with different concentrations of NADPH revealed that the kinetics of lipid peroxidation in lung microsomes was indistinguishable from that of NADPH-dependent lipid peroxidation in liver microsomes. With lung microsomes supplemented with NADPH, lipid peroxidation was potentiated by CCl₄ and inhibited by EDTA, Mn²⁺, and cytochrome c.     

Protective action of acetylcarnitine on NADPH-induced lipid peroxidation of cardiac microsomes

Rat cardiac microsomes treated with NADPH generated a chemiluminescence, detected by the chemilumigenic probe lucigenin. The chemiluminescent signal, which is an index of lipid peroxidation, was found to be inhibited by acetylcarnitine in a dose-dependent way. Superoxide dismutase (SOD) and inhibitors of arachidonate metabolism were also effective in preventing light emission. The combined action of acetylcarnitine plus SOD and acetylcarnitine plus indomethacin suggested a possible common target for the compounds. When tested on superoxide production from isolated human neutrophils detected both by luminol-amplified chemiluminescence and cytochrome C reduction, acetylcarnitine did not show any inhibitory effect. The results of these experiments demonstrate the antioxidant properties of acetylcarnitine, even if they cannot clarify the specific target of the drug action.     

Bromosulfophthalein abolishes glutathione-dependent protection against lipid peroxidation in rat liver mitochondria

The effect of bromosulfophthalein (BSP) on GSH-dependent protection against lipid peroxidation in rat liver mitochondria was examined. Mitochondrial lipid peroxidation induced by ascorbate-Fe2+ was prevented by GSH, and addition of BSP abolished the protective effect of GSH. The effect of BSP was apparently not due to causing disappearance of GSH from the reaction mixture by interacting directly with GSH. BSP strongly inhibited the mitochondrial GSH S-transferase activity rather than the GSH peroxidase activity. Ascorbate-Fe2+-induced lipid peroxidation in mitochondria without addition of GSH was also stimulated to some extent by BSP, and the stimulation seems likely to be due to abolition of the inhibitory effect of endogenous GSH. GSH could not be replaced as an inhibitor of lipid peroxidation by cysteine, beta-mercaptoethanol, or dithiothreitol. The inhibitory effect of GSH on lipid peroxidation was not observed in vitamin E-deficient mitochondria. No inhibitory effect of exogenous vitamin E was demonstrated either in vitamin E-deficient mitochondria or in vitamin E-sufficient mitochondria in the presence of BSP, whether GSH was added or not. These results indicate that a mitochondrial GSH-dependent factor which inhibits lipid peroxidation requires vitamin E to exert its function. It is suggested that mitochondrial GSH S-transferase(s) may be responsible for GSH-dependent inhibition of lipid peroxidation in mitochondria, probably by scavenging lipid radicals.     

Antioxidant effects of albumin-bound sulfur in lipid peroxidation of rat liver microsomes.

The antioxidant potential of albumin-bound sulfur (SBA) was investigated in rat liver microsomes using lipid peroxidation systems in vitro. Sulfur bound to protein is a reduced metabolite which is produced from cystine by gamma-cystathionase. Lipid peroxidation was induced either chemically by ferrous ions and ascorbate or enzymatically by carbon tetrachloride or tert-butyl hydroperoxide as indicated by the increase in thiobarbituric acid reactive substances (TBA-RS) and oxygen consumption. Although the antioxidant effect of SBA was weak on the non-enzymatic lipid peroxidation system, the addition of SBA significantly inhibited TBS-RS formation and oxygen consumption compared with non-treated bovine serum alubumin (BSA) in a microsomal lipid peroxidation system induced enzymatically. The sulfur bound to albumin disappeared during incubation with liver microsomes. However, slight differences in the disappearance were observed depending on whether or not lipid peroxidation was induced in the enzymatic systems. In the CCl4-induced lipid peroxidation system, the cytochrome P-450 level was significantly decreased by the addition of SBA. Therefore, in cytochrome P-450 dependent lipid peroxidation system, the potential effects of sulfur bound to albumin are due to an inhibition of cytochrome P-450 rather than by the oxidation itself caused by radical trapping.     

The role of iron in 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced lipid peroxidation by rat liver microsomes

Treatment of female Sprague-Dawley rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhances hepatic lipid peroxidation, and the role of iron in TCDD-induced lipid peroxidation was examined. Ferrous and ferric ions, and the chelators adenosine diphosphate (ADP), ethylenediaminetetraacetic acid (EDTA) and desferrioxamine (DFX) were added to an in vitro microsomal lipid peroxidation system using microsomes from control and TCDD-treated animals. Both ferrous and ferric ions enhanced microsomal lipid peroxidation, with the greatest effect being produced by the combination of ferrous and ferric ions. The addition of ADP and EDTA produced modest increases in microsomal lipid peroxidation, suggesting that these chelators facilitated formation of reactive oxygen species by iron. The addition of DFX markedly inhibited microsomal lipid peroxidation, with greatest inhibition occurring with microsomes from TCD-treated animals. The results indicate that iron is involved in TCDD-induced lipid peroxidation.     

Inhibitory action of Oren-gedoku-to extract on enzymatic lipid peroxidation in rat liver microsomes

We examined the inhibitory action of the extract of Oren-gedoku-to, a traditional herbal medicine known to act as an antioxidant, on enzymatic lipid peroxidation in rat liver microsomes. Simultaneous addition of a spray-dried preparation of Oren-gedoku-to extract (Tsumura TJ-15) inhibited enzymatic lipid peroxidation induced by reduced beta-nicotinamide adenine dinucleotide phosphate (NADPH) and ADP/Fe3+ complex in liver microsomes in a dose-dependent manner. When the inhibition by TJ-15 of enzymatic lipid peroxidation in liver microsomes was kinetically analyzed, this medicine showed a competitive inhibition against NADPH or ADP/Fe3+ complex. TJ-15 inhibited the NADPH-driven enzymatic reduction of ADP/Fe3+ complex or cytochrome c in liver microsomes competitively. TJ-15 enhanced NADPH consumption by liver microsomes with ADP/Fe3+ complex. Treatment with TJ-15 after the onset of enzymatic lipid peroxidation in liver microsomes inhibited the progression of lipid peroxidation in a dose-dependent manner. The present results indicate that Oren-gedoku-to extract inhibits enzymatic lipid peroxidation in rat liver microsomes in the initiation and propagation steps in a dose-dependent manner. These results also suggest that Oren-gedoku-to extract inhibits enzymatic lipid peroxidation in rat liver microsomes not only through its antioxidant action but also through reduction of the supply of electrons derived from NADPH to ADP/Fe3+ complex in liver microsomes both in a competitive manner and through stimulation of NADPH oxidation.     

Inhibitory action of silymarin of lipid peroxide formation in rat liver mitochondria and microsomes

Silymarin, a 3-oxyflavone present in Silybum Marianum, protected both liver mitochondria and microsomes from lipid peroxide formation induced by various agents. The antiperoxidative action exhibited by Silymarin was 10-fold higher than that of α-tocopherol, and was present when the drug was added as well as after the peroxidant agents. Data obtained rule out the possibility that the antiperoxidative action of Silymarin was due to an interaction of this drug with Fe²⁺. The reported results are compatible with an interaction of Silymarin with free radical species responsible for lipid peroxidation.     

Protection by gsh against lipid peroxidation induced by ascorbate and iron in rat liver microsomes

GSH is considered to be a potent inhibitor of 1ipid peroxidation, but the mechanisms by which it carries out this function are not clear. GSH-dependent factors which inhibit 1ipid peroxidation in the NADPH and in the ascorbate-iron microsomal 1ipid peroxidation systems have been demonstrated in rat liver 105,000 $\text{g}$ supernatant (1,2). This communication describes a GSH-dependent factor in the microsomal fraction of rat liver which inhibits ascorbate and iron-induced microsomal 1ipid peroxidation.     

Effect of multiple administration of calcium antagonists on lipid peroxidation in rat liver microsomes

The effects of three calcium antagonists, nifedipine (NF), verapamil (VP), and diltiazem (DT), on the lipid peroxidation (LPO) in rat liver microsomes were studied. The drugs were administered in oral doses of 50, 40, and 30 mg/kg daily for 21 days in male Wistar rats. Nonstimulated LPO was significantly decreased by NF and was not changed by VP and DT. There was a correlation between the extent of the previously found enzyme-inducing action and the potency of the antioxidant effects of calcium antagonists. Fe²⁺/NADPH- and Fe²⁺/ascorbate-stimulated microsomal LPO was increased by the calcium antagonists studied in the following order: VP>DT>NF (the increase caused by NF was insignificant in Fe²⁺/NADPH stimulation).     

Effects of lipid peroxidation on activities of drug-metabolizing enzymes in liver microsomes of rats

A relationship was found between the formation of lipid peroxides and the activities of drug-metabolizing enzymes in liver microsomes of rats. Induction of lipid peroxidation by incubation with ferrous ion led to a sharp decline in the ability of the microsomal enzyme system to demethylate ethylmorphine. Inhibition of lipid peroxidation by EDTA increased the enzyme activity about two-fold. Addition of EDTA (0.1 mM) to the incubation mixture produced marked changes in the Michaelis constants of drug-metabolizing enzymes and in inhibition constants of SKF 525-A for drug-metabolizing enzymes.     

The effect of diethyldithiocarbamate on the lipid peroxidation of rat-liver microsomes and intact hepatocytes

The role of the oxygen radicals in lipid peroxidation, induced by ADP/Fe³⁺ or cumene hydroperoxide was investigated by administering diethyldithiocarbamate, an inhibitor of Superoxide dismutase, to hepatocytes or rats.Intact rat-liver hepatocytes perform a delayed ADP/Fe³⁺-induced lipid peroxidation after pretreatment with diethyldithiocarbamate. The cumene hydroperoxide-induced lipid peroxidation is unchanged. Hepatocytes, isolated from a rat administered with diethyldithiocarbamate in vivo, exhibit the same pattern, a delayed iron-induced lipid peroxidation and an unchanged cumene hydroperoxide-induced lipid peroxidation.Liver microsomes isolated from liver of a rat administered with diethyldithiocarbamate do not perform lipid peroxidation with NADPH/ADP/Fe³⁺, but do undergo lipid peroxidation with cumene hydroperoxide.It can be concluded that besides the inhibition of Superoxide dimutase, diethyldithiocarbamate inhibits directly the microsomal lipid peroxidation. Although this inhibition hampers the conclusion, evidence is obtained that Superoxide dismutase is probably involved in the protection against lipid peroxidation of the mitochondria, but not of the microsomes.     

NADPH- and linoleic acid hydroperoxide-induced lipid peroxidation and destruction of cytochrome P-450 in hepatic microsomes

Temporal aspects of the effects of inhibitors on hepatic cytochrome P-450 destruction and lipid peroxidation induced by NADPH and linoleic acid hydroperoxide (LAHP) were compared. In the absence of added Fe2+, NADPH-induced lipid peroxidation in hepatic microsomes exhibited a slow phase followed by a fast phase. The addition of Fe2+ eliminated the slow phase, thus demonstrating that iron is a rate-limiting component in the reaction. EDTA, which complexes iron, and p-chloromercurobenzoate (pCMB), which inhibits NADPH-cytochrome P-450 reductase, inhibited both phases of the reaction. Catalase as well as scavengers of hydroxyl radical, inhibited NADPH-induced lipid peroxidation almost completely. GSH also inhibited the NADPH-dependent reaction but only when added at the beginning of the reaction. In contrast with NADPH-dependent lipid peroxidation, the autocatalytic reaction induced by LAHP was not biphasic, NADPH-dependent or iron-dependent, nor was it inhibited by hydroxyl radical scavengers, catalase or GSH. A synergistic effect on lipid peroxidation was observed when both NADPH and LAHP were added to microsomes. It is concluded that both the fast and slow phases of NADPH-dependent microsomal lipid peroxidation are catalyzed enzymatically and are dependent upon Fe2+, whereas LAHP-dependent lipid peroxidation is autocatalytic. Since the fast phase of enzymatic lipid peroxidation occurred during the fast phase of destruction of cytochrome P-450, it is postulated that iron made available from cytochrome P-450 is sufficient to promote optimal lipid peroxidation. Since catalase and hydroxyl radical scavengers inhibited NADPH-dependent but not LAHP-dependent lipid peroxidation, it is concluded that the hydroxyl radical derived from H2O2 is the initiating active-oxygen species in the enzymatic reaction but not in the autocatalytic reaction.