The mechanism of cumene hydroperoxide-dependent lipid peroxidation: The function of cytochrome P-450

doi:10.1016/0003-9861(86)90082-2

Archives of Biochemistry and Biophysics

Volume 251, Issue 1, 15 November 1986, Pages 348-360

https://doi.org/10.1016/0003-9861(86)90082-2 Get rights and content

Abstract

The addition of limiting amounts of cumene hydroperoxide to rat liver microsomes resulted in the rapid uptake of molecular oxygen, the formation of thiobarbituric acid reactive products, and the loss of hydroperoxide. The stoichiometry of lipid peroxidation and the yields of 2-phenyl-2-propanol (a major product of the reaction) and acetophenone (a minor product) observed with liver microsomes prepared from untreated rats is greater than that seen with liver microsomes from ciprofibrate-treated rats which, in turn, is greater than that observed with liver microsomes from phenobarbital-treated rats. The K_m's and V_max's of oxygen uptake varied with the type of rat liver microsomes used. Cytochrome P-450 substrates and inhibitors decreased the extents and initial rates of oxygen uptake and thiobarbituric acid reactive product formation. A mechanism is proposed involving the cytochrome P-450-catalyzed homolytic cleavage of the cumene hydroperoxide OO bond to give the cumyloxyl radical. It is proposed that this oxygen-centered radical abstracts a hydrogen atom from an unsaturated fatty acid associated with a lipid (initiating lipid peroxidation) to give 2-phenyl-2-propanol or that the radical undergoes β-scission to produce acetophenone and a methyl radical.

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Mechanism of action of novel naphthofuranquinones on rat liver microsomal peroxidation
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Citation Excerpt :
The reaction was started by adding ascorbate, which was prepared immediately before use and kept under a stream of nitrogen. For the t-BuOOH-Fe induced peroxidation [26], the incubation mixture consisted of 0.11 mM EDTA, 0.10 mM FeSO4, 2.6 mM t-BuOOH, microsomes (1.5 mg protein/ml) and phosphate buffer as with the NADPH-generating system. EDTA and FeSO4 were added as an EDTA–FeSO4 complex, which was prepared immediately before use and kept under a stream of nitrogen; then, the reaction was started by adding t-BuOOH.
In order to elucidate the effect on mammal systems of new derivatives from 2-hydroxy-3-allyl-naphthoquinone, α-iodinated naphthofuranquinone (NPPN-3223), β-iodinated naphthofuranquinone (NPPN-3222) and β-methyl naphthofuranquinone (NPPN-3226) synthesized as possible trypanocidal agents, their effect on rat liver microsomal lipid peroxidation was investigated. They (a) inhibited NADPH-dependent, iron-catalyzed microsomal rat liver lipid peroxidation; (b) did not inhibit the tert-butyl hydroperoxide-dependent lipid peroxidation; (c) did not inhibit ascorbate–lipid peroxidation with the exception of NPPN-3226 which did inhibit it; (d) stimulated NADPH oxidation and microsomal oxygen uptake; (e) increased superoxide anion formation by NADPH-supplemented microsomes and (f) stimulated ascorbate oxidation. The three drugs were reduced to their seminaphthofuranquinone radical by the liver NADPH–P450 reductase system, as detected by ESR measurements. These results support the hypothesis that naphthofuranquinones reduction by microsomal NADPH–P450 reductase and semiquinone oxidation by molecular oxygen diverts electrons, preventing microsomal lipid peroxidation. In addition, hydroquinones and/or semiquinones formed by naphthofuranquinones reduction would be capable of lipid peroxidation inhibition and on interacting with the lipid peroxide radicals can lead to an antioxidant effect as we suggested for NPPN-3226 in close agreement to the inhibition of ascorbate–lipid peroxidation. Due to the properties of these molecules and their incoming structure developments, naphthofuranquinones would be considered as potentially promising therapeutic agents, mainly against Chagas disease.
Protection of cells from oxidative stress by microsomal glutathione transferase 1
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By gating to eliminate background fluorescence from unexposed cells, antisense cells but not sense cells exposed to 50 μM CuOOH showed an elevated level of intracellular ROS (Fig. 3B), indicating that MGST1 was able to eliminate ROS generated from LPO-induced oxidative stress. Two hydroperoxides, CuOOH and H2O2, a substrate, and non-substrate, which differed in lipid solubility and efficiency to induce LPO in the rat liver microsomal fraction [18,22], were employed to treat sense and antisense cells. Results demonstrated that MGST1 protected cells from toxicity of both CuOOH and H2O2 as determined by both MTT and LDH assays.
Rat liver microsomal glutathione transferase 1 (MGST1) is a membrane-bound enzyme that displays both glutathione transferase and glutathione peroxidase activities. We hypothesized that physiologically relevant levels of MGST1 is able to protect cells from oxidative damage by lowering intracellular hydroperoxide levels. Such a role of MGST1 was studied in human MCF7 cell line transfected with rat liver mgst1 (sense cell) and with antisense mgst1 (antisense cell). Cytotoxicities of two hydroperoxides (cumene hydroperoxide (CuOOH) and hydrogen peroxide) were determined in both cell types using short-term and long-term cytotoxicity assays. MGST1 significantly protected against CuOOH and against hydrogen peroxide (although less pronounced and only in short-term tests). These results demonstrate that MGST1 can protect cells from both lipophilic and hydrophilic hydroperoxides, of which only the former is a substrate. After CuOOH exposure MGST1 significantly lowered intracellular ROS as determined by FACS analysis.
Formation of DNA adducts by microsomal and peroxidase activation of p-cresol: Role of quinone methide in DNA adduct formation
2001, Chemico-Biological Interactions
Citation Excerpt :
The DNA adducts detected by HRP activation may occur from reaction of either the semiquinone or the quinone methide with DNA. Cytochrome P450 in the presence of CuOOH as a cofactor has been shown to initiate radical reactions in addition to oxygenation [34–37]. Therefore with CuOOH as the cofactor p-cresol may be oxidized to a phenoxy radical similar to that formed during HRP activation.
We have investigated the activation of p-cresol to form DNA adducts using horseradish peroxidase, rat liver microsomes and MnO₂. In vitro activation of p-cresol with horseradish peroxidase produced six DNA adducts with a relative adduct level of 8.03±0.43×10⁻⁷. The formation of DNA adducts by oxidation of p-cresol with horseradish peroxidase was inhibited 65 and 95% by the addition of either 250 or 500 μM ascorbic acid to the incubation. Activation of p-cresol with phenobarbital-induced rat liver microsomes with NADPH as the cofactor; resulted in the formation of a single DNA adduct with a relative adduct level of 0.28±0.08×10⁻⁷. Similar incubations of p-cresol with microsomes and cumene hydroperoxide yielded three DNA adducts with a relative adduct level of 0.35±0.03×10⁻⁷. p-Cresol was oxidized with MnO₂ to a quinone methide. Reaction of p-cresol (QM) with DNA produced five major adducts and a relative adduct level of 20.38±1.16×10⁻⁷. DNA adducts 1, 2 and 3 formed by activation of p-cresol with either horseradish peroxidase or microsomes, are the same as that produced by p-cresol (QM). This observation suggests that p-cresol is activated to a quinone methide intermediate by these activation systems. Incubation of deoxyguanosine-3′-phosphate with p-cresol (QM) resulted in a adduct pattern similar to that observed with DNA; suggesting that guanine is the principal site for modification. Taken together these results demonstrate that oxidation of p-cresol to the quinone methide intermediate results in the formation of DNA adducts. We propose that the DNA adducts formed by p-cresol may be used as molecular biomarkers of occupational exposure to toluene.
The antioxidant action of 2-methyl-6-(p-methoxyphenyl)-3,7- dihydroimidazo[1,2-α]pyrazin-3-one (MCLA), a chemiluminescence probe to detect superoxide anions
1998, FEBS Letters
The antioxidant effect of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α]pyrazin-3-one (MCLA), a Cypridina luciferin analog that acts as a chemiluminescence probe to detect O^⋅−₂, was investigated. MCLA produced a lag in oxygen consumption induced by cumene hydroperoxide in microsomes or by 2,2′-azobis (2-amidinopropane) dihydrochloride in liposomes and disappeared during the duration of the lag. MCLA profoundly inhibited the propagation reaction in Fe²⁺-dependent lipid peroxidation in liposomes, and MCLA disappearance accompanied by suppression of oxygen consumption markedly occurred in liposomes susceptible to peroxidation. Thiobarbituric acid-reactive substances in all systems used were also suppressed by MCLA dose dependently. These results indicate that MCLA has an antioxidant property through scavenging free radicals.
Inhibitory effect of free radicals derived from organic hydroperoxide on progesterone synthesis in human term placental mitochondria
1998, Free Radical Biology and Medicine
Different natural and synthetic organic hydroperoxides have been found to stimulate TBARS formation in human term placental mitochondria. The levels of TBARS were lower than arising from NADPH-dependent lipid peroxidation. BHT, Mn²⁺ and DMPO counteracted TBARS formation in the presence of cumene hydroperoxide implicating involvement of free radicals in this process. On the other hand superoxide dismutase, catalase and EDTA while being inhibitory in NADPH-dependent lipid peroxidation did not inhibit cumene hydroperoxide-dependent TBARS formation. Amphenone B and SKF-525A, inhibitors of cytochrome P-450, strongly inhibit both NADPH- and cumene hydroperoxide-dependent lipid peroxidation. These data provide evidence that cytochrome P-450_SCC is involved in both these processes. However NADPH-dependent lipid peroxidation and the cumene hydroperoxide have been found to inactivate placental mitochondrial cytochrome P-450_SCC. The presence of cumene hydroperoxide resulted in a more rapid inactivation of cytochrome P-450_SCC and consequently inhibited NADPH-dependent lipid peroxidation. It has been observed for the first time that progesterone biosynthesis can be inhibited by cumene hydroperoxide. Protective effect of Mn²⁺ and DMPO on progesterone biosynthesis indicates the importance of free radicals as transient products of cytochrome P-450_SCC-dependent cumene hydroperoxide metabolism. In contrast to progesterone formation from cholesterol, the conversion of pregnenolone to progesterone was not affected by cumene hydroperoxide. This suggests that inhibition of progesterone synthesis from cholesterol by hydroperoxide may be ascribed to its effect on the desmolase activity of cytochrome P-450_SCC in placental mitochondria. On the basis of the results obtained, we propose that the inhibition of progesterone biosynthesis by naturally occurring hydroperoxides may contribute to the development of preeclampsia.
Enzymatic and molecular aspects of the antioxidant effect of menadione in hepatic microsomes
1996, Archives of Biochemistry and Biophysics
The enzymatic features and molecular species of the inhibitory action of menadione on lipid peroxidation in rat liver microsomes were examined. In an ascorbate-supported system or a NADH-supported reconstituted system containing NADH–cytochrome b₅reductase and cytochrome b₅, menadione was not an inhibitor of lipid peroxidation at pH 7.5, while some antioxidant ability was observed at lower pH ranges. Lipid peroxidation in the presence of menadione in the NADH-supported reconstituted system at pH 7.5 was markedly inhibited by adding lipoamide dehydrogenase. NAD(P)H-supported lipid peroxidation in microsomes with increased DT-diaphorase activity from 3-methylcholanthrene-treated rats was highly susceptible to menadione. These inhibitions were abolished by dicoumarol, an inhibitor of DT-diaphorase. Cumene hydroperoxide-dependent lipid peroxidation in microsomes, with desferal and NADP⁺to prevent nonheme iron-dependent reactions and oxygen radical generation, was inhibited by menadione in the presence of NADPH, and the inhibition was also more effective in the microsomes with increased DT-diaphorase activity. Menadiol reacted with 1,1-diphenyl-2-picrylhydrazyl (DPPH) in ethanol at a molar ratio of DPPH/menadiol at 1.9. In an iron-supported reconstituted enzymatic or a nonenzymatic system at pH 7.5, menadiol showed an antioxidant effect at an early stage, followed by a prooxidant effect, which was prevented by SOD, probably by protecting menadiol autooxidation. These results show that menadione exerts an antioxidant effect through participation of microsomal DT-diaphorase by generating menadiol with a radical scavenging ability, while menadiol also has a prooxidant property.

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^☆: Supported in part by grants from the U.S. Public Health Service National Institutes of Health (NIGMS 16488) and The Robert A. Welch Foundation (I-959).

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The mechanism of cumene hydroperoxide-dependent lipid peroxidation: The function of cytochrome P-450☆

Abstract

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