Detection of free radicals produced from the reaction of cytochrome P-450 with linoleic acid hydroperoxide

Rota, Cristina; Barr, David P.; Martin, Martha V.; Guengerich, F. Peter; Tomasi, Aldo; Mason, Ronald P.

doi:10.1042/bj3280565

Cited by 59 publications

(49 citation statements)

References 50 publications

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“…This is followed by radical rebound resulting in the formation and release of the alcohol metabolite (Atkinson and Ingold, 1993; dmd.aspetjournals.org Shaik et al, 2002). The carbon-centered radical is very short-lived and is rapidly hydroxylated and released from the active site as a metabolite much faster than the radical itself can escape from the hydrophobic enzyme active site (Heur et al, 1989;Bowry et al, 1990;Rota et al, 1997). This scenario reflects the metabolism of PCP by P450 2B6 and accounts for the partition ratio for P450 2B6 inactivation by PCP as being 45 (Jushchyshyn et al, 2003), which suggests that the majority of PCP secondary carbons are oxidized and released from the active site as hydroxylated metabolites (M1 and M2).…”

Section: Discussionmentioning

confidence: 99%

Mechanism of Inactivation of Human Cytochrome P450 2B6 by Phencyclidine

Jushchyshyn¹,

Wahlstrom

Hollenberg³

et al. 2006

Drug Metabolism and Disposition

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ABSTRACT:The mechanism behind the observed inactivation of human P450 2B6 by phencyclidine (PCP) has been evaluated over the past 2 decades. The scope of the current investigation was to contribute to the fundamental knowledge of PCP oxidation and perhaps the mechanism behind P450 inactivation. To study the chemistry of PCP oxidation, we subjected PCP to the Fenton reagent. Under Fenton chemistry conditions, oxidation on all three PCP rings was observed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). When PCP was incubated with the Fenton system in the presence of glutathione (GSH), three GSH-PCP conjugates were identified. Subsequent LC-MS/MS analysis of these conjugates revealed two species that had GSH attached to the cyclohexane ring of PCP and a third conjugate in which GSH was adducted to the piperidine ring. When PCP was incubated across a panel of P450 enzymes, several enzymes, including P450s 2D6 and 3A4, were able to catalyze the formation of the PCP iminium ion, whereas P450s 2B6 and 2C19 were exclusively able to hydroxylate secondary carbons on the cyclohexane ring of PCP. Subsequent mechanistic experiments revealed that only P450s 2B6 and 2C19 demonstrated loss of catalytic activity after preincubation with 10 M PCP. Finally, investigation of P450 2B6 inactivation using structural analogs of PCP revealed that blocking the para-carbon atom on the cyclohexane ring of PCP from oxidation protected the P450 2B6 from inactivation, which suggests that a reactive intermediate generated during the hydroxylation of the cyclohexane ring may be linked to the mechanism of inactivation of P450 2B6 by PCP.The biotransformation of phencyclidine, PCP (Fig. 1), by liver proteins was shown to result in the formation of reactive metabolites leading to P450 inactivation and the formation of covalent adducts with hepatic macromolecules more than 20 years ago (Castagnoli et al., 1997). Originally, covalent binding and enzyme inactivation was thought to occur via alkylation of protein-based nucleophiles by an iminium ion metabolite, M4 (Fig. 1), that arose from the P450-dependent two-electron ␣-carbon oxidation of PCP (Sayre et al., 1997). This hypothesis was supported by the formation of PCP iminium-cyanide adducts and the observation that the presence of cyanide in microsomal incubations protected against the formation of covalent adducts with proteins (Ward et al., 1982b;Hoag et al., 1984). However, subsequent studies demonstrated that the P450 inactivation by the PCP iminium ion (PCP-Im) required the presence of NADPH (Hoag et al., 1984;Osawa and Coon, 1989;Sayre et al., 1991), which inferred that metabolism of PCP beyond the iminium species was required for P450 inactivation.Later, the PCP mechanism-based inactivation of P450 2B6, using purified, recombinant human P450 enzyme, was investigated in great detail (Jushchyshyn et al., 2003). Through a series of experiments, it was shown that P450 2B6 was inactivated via covalent modification of the P450 2B6 apoprotein. Moreover, inclusion of cyanide in the pu...

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Section: Discussionmentioning

confidence: 99%

Mechanism of Inactivation of Human Cytochrome P450 2B6 by Phencyclidine

Jushchyshyn¹,

Wahlstrom

Hollenberg³

et al. 2006

Drug Metabolism and Disposition

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“…To date, nitrone spin-trapping agents, including ␣ -[4-pyridyl-1-oxide] N -tert-butyl nitrone and 5,5 Ј -dimethyl-1-pyroline-N -oxide, have been used to detect these lipid-derived radicals (12)(13)(14)(15)(16)(17). Furthermore, Mason and coworkers (13)(14)(15) established an HPLC/ESR system to identify the resulting radical adducts.…”

mentioning

confidence: 99%

Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition

Koshiishi

Tsuchida

Takajo

et al. 2005

Journal of Lipid Research

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Lipid alkyl radicals generated from polyunsaturated fatty acids via chemical or enzymatic H-abstraction have been a pathologically important target to quantify. In the present study, we established a novel method for the quantification of lipid alkyl radicals via nitroxyl radical spintrapping. These labile lipid alkyl radicals were converted into nitroxyl radical-lipid alkyl radical adducts using 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N -oxyl (Cm ⌬ P) (a partition coefficient between octanol and water is approximately 3) as a spin-trapping agent. The resulting Cm ⌬ P-lipid alkyl radical adducts were determined by HPLC with postcolumn online thermal decomposition, in which the adducts were degraded into nitroxyl radicals by heating at 100 ؇ C for 2 min. The resulting nitroxyl radicals were selectively and sensitively detected by electrochemical detection. With the present method, we, for the first time, determined the lipid alkyl radicals generated from linoleic acid, linolenic acid, and arachidonic acid via soybean lipoxygenase-1 or the radical initiator 2,2 -azobis(2,4-dimethyl-valeronitrile). Peroxidation of polyunsaturated fatty acids, which are components of cellular membrane and lipoproteins, leads to vital damage to several types of cells. Reactive oxygen/nitrogen species (1), such as hydroxyl radical, hypochlorous acid (2-4), peroxynitrite (5, 6), and singlet oxygen molecule (7-9) are involved in the peroxidation of polyunsaturated fatty acids in the inflammatory lesions. On the other hand, lipoxygenases are speculated to be involved in the peroxidation of polyunsaturated fatty acids in the atheromatous plaque (10). Commonly, lipid alkyl radicals seem to be necessarily produced as an intermediate in these reactions.To detect labile free radicals via ESR, spin-trapping techniques have been established, in which unstable radicals react with spin-trapping agents to form relatively stable radical adducts (11). To date, nitrone spin-trapping agents, including ␣ -[4-pyridyl-1-oxide] N -tert-butyl nitrone and 5,5 Ј -dimethyl-1-pyroline-N -oxide, have been used to detect these lipid-derived radicals (12-17). Furthermore, Mason and coworkers (13-15) established an HPLC/ESR system to identify the resulting radical adducts. However, the trapping efficiency of these nitrone compounds toward lipid alkyl radicals is comparatively low. Consequently, these spin-trapping agents are not applicable to the quantification of lipid alkyl radicals. Five-or six-membered cyclic nitroxyl radicals are relatively stable. Nitroxyl radicals appeared to possess an ability to scavenge carbon-centered radicals (18). This special character of nitroxyl radicals makes it possible to quantitatively trap carbon-centered radicals. Johnson, Caron, and Blough (19) used hydrophilic 3-aminomethyl-2,2,5,5-tetramethylpyrrolidine-N -oxyl to evaluate carbon-centered radical generation: in this method, the adducts were fluorometrically detected after derivatization of amino groups by fluorescamine.The nitroxyl radical-carbon-centered radi...

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“…In this study, the 4-POBN\octanoic acid and 4-POBN\pentyl radical adducts were detected and identified in the reaction mixture of 13-HPODE with cytochrome c or haematin using HPLC-ESR and HPLC-ESR-MS. Our previous studies have also shown the formation of the octanoic acid and pentyl radicals [23] and Rota et al [10] have also presented homolytic scission for the reaction of cytochrome c with tbutylhydroperoxide [23] and the reaction of cytochrome P450 with 13-HPODE [10]. On the other hand, it is also possible for heterolytic peroxide bond cleavage to result in the formation of the alkoxyl radical, as follows [24][25][26] :…”

Section: Discussionmentioning

confidence: 99%

Cytochrome c catalyses the formation of pentyl radical and octanoic acid radical from linoleic acid hydroperoxide

Iwahashi

Nishizaki

Takagi

2001

Biochemical Journal

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A reaction of 13-hydroperoxide octadecadienoic acid (13-HPODE) with cytochrome c was analysed using ESR, HPLC–ESR and HPLC–ESR–MS by the combined use of the spin-trapping technique. The ESR, HPLC–ESR and HPLC–ESR–MS analyses showed that cytochrome c catalyses formation of pentyl and octanoic acid radicals from 13-HPODE. On the other hand, only the α-(4-pyridyl-1-oxide)-N-t-butylnitrone/octanoic acid radical adduct was detected in the elution profile of HPLC–ESR for a mixture of 13-HPODE with haematin, indicating that haematin catalyses the formation of octanoic acid radical. In addition, the reaction of 13-HPODE with cytochrome c was inhibited by chlorogenic acid, caffeic acid and ferulic acid via two possible mechanisms, i.e. reducing cytochrome c (chlorogenic acid and caffeic acid) and scavenging the radical intermediates (chlorogenic acid, caffeic acid and ferulic acid).

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Detection of free radicals produced from the reaction of cytochrome P-450 with linoleic acid hydroperoxide

Cited by 59 publications

References 50 publications

Mechanism of Inactivation of Human Cytochrome P450 2B6 by Phencyclidine

Mechanism of Inactivation of Human Cytochrome P450 2B6 by Phencyclidine

Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition

Cytochrome c catalyses the formation of pentyl radical and octanoic acid radical from linoleic acid hydroperoxide

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