Liver microsomal cytochrome P-450 and the oxidative metabolism of arachidonic acid.

Abstract

Arachidonic acid is oxidatively metabolized by rat liver microsomes at a rate of approximately 5 nmol per min per mg of protein at 25C. This reaction is dependent on the presence of NADPH and oxygen. Studies with various inhibitors indicate a role for membrane-bound cytochrome P-450 in the transformation of arachidonic acid to a mixture of hydroxy acid derivatives. The stoichiometry of the reaction conforms to that of a monooxygenase reaction-i.e., one mole of NADPH is oxidized per mole of oxygen utilized-suggesting a reaction mechanism different from that proposed for lipid peroxidation reactions. No evidence for the formation of prostaglandin-like metabolites was obtained. The diene character of some of the metabolites formed suggests another role for cytochrome P-450-i.e., participation in hydrogen abstraction reactions for the activation of various substrates.The oxidative metabolism ofarachidonic acid can lead to a wide variety of metabolites. Interest in the physiological function of prostaglandins, thromboxanes, leukotrienes, and other oxidative metabolites of arachidonic acid, as well as the reactions of lipid peroxidation, have established the need to better understand the role of the various enzymes responsible for oxygen incorporation during the process of arachidonic acid metabolism-e.g., cyclooxygenase, lipoxygenase, etc. (1, 2).The microsomal fraction from rat liver, like that from many other tissues, is rich in phospholipids containing arachidonic acid (3). During the NADPH-and oxygen-dependent function of the liver microsomal cytochrome P-450-containing electron transport system, a significant portion of electrons is diverted to either the formation of hydrogen peroxide or the oxidative transformation of "endogenous substrates" (4, 5). The latter reaction conforms to the stoichiometry of a monooxygenase reaction in that one mole of NADPH is oxidized for each mole of oxygen consumed. The nature of the endogenous substrate(s) of liver microsomes has eluded characterization, although Schenkman et al. (6) have provided evidence that unsaturated fatty acids, in particular oleic acid, might serve in this capacity. In addition, it has been reported that unsaturated fatty acids appear to be released from liver microsomal phospholipids during the course of NADPH oxidation (7,8). In the presence of an iron chelate, such as the ADP-Fe3+ complex, and an electron donor, such as NADPH or ascorbate, malonaldehyde is rapidly formed concomitant with a rapid rate of oxygen utilization (9). It is generally assumed that one source of malonaldehyde is from the oxidation of arachidonic acid (1, 10). Thus, it was of interest to evaluate the oxidative metabolism ofarachidonic acid by the electron transport system associated with rat liver microsomes.It is the purpose of this communication to describe the role of liver microsomal cytochrome P-450 in the oxidation of arachidonic acid. It is suggested that free arachidonic acid, as well as that derived from microsomal phospholipids, may contribute, in part, to the...

Mitochondrial P450-dependent arachidonic acid metabolism by TCDD-induced hepatic CYP1A5; conversion of EETs to DHETs by mitochondrial soluble epoxide hydrolase

“…Although the liver is a well recognized site of P450 aa metabolism [21,42] and P450 aa metabolism was first identified in liver microsomes [43], effects of P450 aa metabolites in liver physiology and pathophysiology have not yet been delineated. Effects of EETs on liver cells or liver microsomes include increased intracellular Ca 2+ , vasopressin stimulated glycogenolysis, activation of phosphorylase and ADP ribosylation (reviewed in Spector and Norris [16]).…”

“…Although human CYP1A2 and avian CYP1A5 (more than other mammalian CYP1A2 enzymes [12,57]) have been demonstrated to have epoxygenase activities, and CYP1A induction was discovered to enhance aa epoxygenation early in the history of P450 mediated aa metabolism [43], attention to CYP1A epoxygenase activity has been eclipsed by interest in constitutive aa epoxygenases such as CYP2C and 2J, P450s, present in vascular endothelial and smooth muscle cells or heart [19]. Several factors should serve to increase attention to CYP1A2/1A5 mediated aa metabolism: (1) CYP1A2 is induced not only in liver, but in kidney, a well recognized site of action for P450-dependent aa metabolites [19,58], (2) CYP1A enzymes are induced not only by toxic chemicals but also by many aryl hydrocarbon receptor ligands such as tryptophan derivatives and other substances in food, as well as some endogenous CYP1A substrates, i.e.…”

Inhibition of arachidonic acid metabolism and its implication on cell proliferation and tumour-angiogenesis

“…The corresponding metabolites include a family of lipoxygenase-like HETEs, epoxyeicosatrienoic acids (EETs) and ω-HETEs which are formed by bis-allylic monoxygenation, olefin epoxydation and ω-hydroxylation respectively [85]. In addition, the CYP450 pathway gives rise to ROS termed HpETEs, although the EETs and ω-HETEs are the major products of the CYP450 pathway [86].…”

Reactive oxygen species and cellular oxygen sensing

“…A high concentration of mitochondrial SOD ensures no significant O 2 .− accumulation beyond its initial site of production and allows for the generation of H 2 O 2 which has second messenger effects in the cytosol [31,[38][39][40]. The endoplasmic reticulum (ER) is another cellular source of ROS where resident cytochrome P-450 and b 5 family members oxidize unsaturated fatty acids and xenobiotics to generate O 2 .− and H 2 O 2 [41][42][43]. In addition, plasma membrane-associated oxidases such as NADPH oxidases generate ROS by oxidizing intracellular NADPH to reduce O 2 into O 2 .− in order to achieve localized microbicidal function in phagosomes [44][45][46][47].…”