Arachidonic acid is an omega-6 polyunsaturated long chain fatty acid that contains 20 carbon atoms and four cis
-double bonds and possesses a carboxyl group and a methyl group at respective ends of the molecule. The double bonds are located between carbons 5-6, 8-9, 11-12, and 14-15 relative to the carboxyl group. Therefore, its chemical name is all-cis
-5,8,11,14-eicosatetraenoic acid and its lipid name is 20
4 (n-6). The cis
-configuration of the four double bonds causes the arachidonic acid backbone to significantly bend. In contrast, double bonds in the trans
-configuration or saturated arachidonic acid result in structurally unbent or flexible backbones.
Experiments performed more than 30 years ago showed that incubations of radio-labelled arachidonic acid with microsomal preparations derived from a variety of tissues including liver [5
], kidney [7
], hypothalamus [8
], and anterior pituitary [9
] resulted in the formation of EETs. This “epoxygenase” reaction required cytochrome p450 (CYP) enzymes and utilised NADPH and oxygen in a 1
1 stoichiometric ratio [5
]. One atom of molecular oxygen is incorporated into one of the four double bonds of arachidonic acid retaining the cis
-geometry and yielding four potential EETs, that is, 5,6-EET, 8,9-EET, 11,12-EET, or 14,15-EET, respectively. Furthermore, each EET can be present in either the S/R or R/S stereoconfiguration, thus eight potential EETs can be formed.
2.1. Epoxygenation of Arachidonic Acid Performed by Specific CYPs
CYP enzymes catalyze the oxidation of organic substances, as well as xenobiotics. Altogether, 57 putative CYP genes have been identified in man (by comparison mice have 103 and rat 89 CYP, resp.) that are divided into 15 subfamilies [10
]. Attempts have been made to classify human CYP genes by substrate; however, a more systematic nomenclature is generally used since the true physiological roles of many of these genes are still unknown [11
]. To date, at least 12 human CYP genes have been reported to possess epoxygenase activity, although most studies have been focussed on the CYP2C and CYP2J families, which are considered the major epoxygenase enzymes.
One of the earliest studies using recombinant human CYP compared the metabolic profiles of the CYP2C8 and CYP2C9 enzymes [12
], which are 77% identical at the amino acid level. Despite their high similarity, CYP2C8 and CYP2C9 exhibit both regio- and stereoselective differences in their epoxygenation of arachidonic acid. For instance, CYP2C8 produced only 14,15-EET and 11,12-EET at a 1.25
1 ratio, which represented 68% of the total metabolites measured. By contrast, CYP2C9 produced 14,15-EET 11,12-EET and 8,9-EET at a ratio of 2.3
0.5, which represented 69% of total metabolites. Furthermore, with respect to stereoselectivity, CYP2C8 was 81% selective for the 11(R),12(S)-EET configuration, whereas CYP2C9 was 70% selective for the 11(S),12(R)-EET configuration [12
]. These CYP enzymes also carry out other reactions including allylic hydroxylation on arachidonic acid and other fatty acids.
Epoxygenase activity of human CYP2J2 was first demonstrated by the Zeldin lab, who initially cloned and characterised the gene [13
]. Recombinant CYP2J2 metabolised arachidonic acid to all four potential epoxygenase products, with 14,15-EET being the predominant metabolite formed. CYP2J2 was found to be highly expressed in the heart, and EETs were produced in similar proportions as recombinant CYP2J2 suggesting that CYP2J2 played a major role in EET generation in the heart in vivo
]. CYP2J2 expression is also seen in kidney, liver and muscle tissues [13
], and, to a lesser extent, in the gut [14
2.4. Other CYPs
A comprehensive comparison study by Rifkind and colleagues examined the epoxygenase activity of a panel of 10 CYP proteins by overexpressing them in HepG2 cells and measuring metabolic products. CYP 2C8, 2C9, 1A2, and 2E1 principally produced epoxygenase products. By contrast, CYP2D6 was inactive, while CYPs 2A6, 3A3, 3A4, and 3A5 had minimal epoxygenase activity [15
]. CYP3A4 has also been shown to make the epoxygenase products 8,9-EET, 11,12-EET, and 14,15-EET, respectively, in several breast cancer cell lines [16
]. Other CYPs that have been shown to possess epoxygenase activity include CYP1A, CYP2B1 and CYP2B2 [17
] and CYP2B12 [18
], CYP2C8, CYP2C9, CYP2D18 [19
], CYP2N1 and CYP2N2 [20
], and rat CYP4A2 and 4A3 [21
]. The full extent of the epoxygenase activity of these enzymes and the physiological consequences of any activity is, however, poorly understood.