Human CYP4F3s are the main catalysts in the oxidation of fatty acid epoxides

Abstract

CYP4F isoforms are involved in the oxidation of important cellular mediators such as leukotriene B 4 (LTB4) and prostaglandins. The proinflammatory agent LTB4 and cytotoxic leukotoxins have been associated with several inflammatory diseases. We present evidence that the hydroxylation of Z 9(10)-epoxyoctadecanoic, Z 9(10)-epoxyoctadec-Z 12-enoic, and Z 12(13)-epoxyoctadec-Z 9-enoic acids and that of monoepoxides from arachidonic acid [epoxyeicosatrienoic acid (EET)] is important in the regulation of leukotoxin and EET activity. These three epoxidized derivatives from the C18 family (C18-epoxides) were converted to 18-hydroxy-C18-epoxides by human hepatic microsomes with apparent K m values of between 27.6 and 175 M. Among recombinant P450 enzymes, CYP4F2 and CYP4F3B catalyzed mainly the -hydroxylation of C18-epoxides with an apparent V max of between 0.84 and 15.0 min ؊ 1 , whereas the apparent V max displayed by CYP4F3A, the isoform found in leukocytes, ranged from 3.0 to 21.2 min ؊ 1 . The rate of -hydroxylation by CYP4A11 was experimentally found to be between 0.3 and 2.7 min ؊ 1 . CYP4F2 and CYP4F3 exhibited preferences for -hydroxylation of Z 8(9)-EET, whereas human liver microsomes preferred Z 11(12)-EET and, to a lesser extent, Z 8(9)-EET. Moreover, vicinal diol from both C18-epoxides and EETs were -hydroxylated by liver microsomes and by CYP4F2 and CYP4F3. These data support the hypothesis that the human CYP4F subfamily is involved in the -hydroxylation of fatty acid epoxides. These findings demonstrate that another pathway besides conversion to vicinal diol or chain shortening by ␤ -oxidation exists for fatty acid epoxide inactivation. -Le Quéré, V., E. Plée-Gautier, P. Potin, S. Madec, and J-P. Salaün. Human CYP4F3s are the main catalysts in the oxidation of fatty acid epoxides.

CYP4F3B Expression Is Associated with Differentiation of HepaRG Human Hepatocytes and Unaffected by Fatty Acid Overload

“…It is noteworthy that the same RP-HPLC profile of radiolabeled metabolites was observed in both extracts: 9,10-dihydroxystearic acid (peak 2), 18-hydroxy-9,10-epoxystearic acid (peak 5), and a low amount of 9,10,18-trihydroxystearic acid (peak 4) were generated. Note that this RP-HPLC profile was similar to that previously obtained after incubation of human liver microsomes with Z9(10)-EpSTA (Le Quéré et al, 2004). The chemical structure of these metabolites was confirmed by GC-MS analysis (data not shown).…”

“…The catalytic functionality of CYP4F3B was assessed by incubating for 30 min differentiated HepaRG cells with [1-14 C]Z9(10)-EpSTA, a discriminating CYP4F3B substrate (Le Quéré et al, 2004). Measurement of the residual radioactivity of the medium and the cell pellet showed a disappearance of more than 30% of the starting radioactivity, suggesting an efficient decarboxylation of the substrate probably by ␤-oxidation.…”

“…GC-MS analysis (electron ionization, 70 eV) was carried out on a gas chromatograph (HP 5890 Series II; Agilent Technologies) equipped with a fused silica capillary column (HP-5MS 5% phenyl methyl siloxane; 30 m ϫ 0.32 mm i.d., 0.25-m thick film) as described previously (Le Quéré et al, 2004). Mass spectra of Me/methyl ester trimethylsilyl ether derivatives contained in peaks with retention times of 21.2 and 26.5 min showed typical fragment ions at m/z 73, 113, 155, 215, 259, and 443 (M ϩ -31) corresponding to 9,10-dihydroxyoctadecanoic acid (see Fig.…”

“…Differentiated HepaRG cells were incubated with 20 M radiolabeled [1-14 C]Z9-(10)-epoxystearic acid (specific activity, 4 Ci/mol), a discriminating substrate of CYP4F3 (Le Quéré et al, 2004), for 30 min at 37°C. Z9-(10)-EpSTA was added in DMSO to 5 ml of culture medium.…”

CYP4A11 is repressed by retinoic acid in human liver cells

“…Since CYP4A11 is the main x-LAH in liver, this duality of effects is likely involved in control of lipid levels in human liver. Its role is still poorly understood since, in humans, it metabolises endogenous substrates such as medium-or long-chain fatty acids, but not arachidonic acid [32]. Instead of acting through catabolic processes, CYP4A11 could generate the medium-and long-chain x-hydroxylated fatty acids endowed with various biological properties.…”