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OtherPROSTAGLANDINS, LEUKOTRIENES AND OTHER EICOSANOIDS

Cytochromes P450 with Bisallylic Hydroxylation Activity on Arachidonic and Linoleic Acids Studied with Human Recombinant Enzymes and with Human and Rat Liver Microsomes

Johan Bylund, Tina Kunz, Karin Valmsen and Ernst H. Oliw
Journal of Pharmacology and Experimental Therapeutics January 1998, 284 (1) 51-60;

Abstract

Bisallylic carbons of polyunsaturated fatty acids can be hydroxylated in NADPH-dependent reactions in liver microsomes. Human recombinant cytochromes P450 and human and rat liver microsomes were assayed for bisallylic hydroxylation activity. CYP1A2, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 converted [14C]linoleic acid to14C-labeled 11-hydroxyoctadecadienoic acid (11-HODE), whereas [14C]arachidonic acid was oxygenated by CYP1A2 and CYP3A4 to 14C-labeled 13-hydroxyeicosatrienoic acid (13-HETE), 10-HETE and 7-HETE as determined by HPLC. Both substrates were also converted to many other metabolites. CYP2C9 appeared to form 12R-HETE and 13-HETE, whereas CYP2C8 formed 13-HETE, 11-HETE and 15-HETE as main monohydroxy metabolites. Fetal human liver microsomes metabolized linoleic acid to 11-HODE as a major hydroxy metabolite, whereas arachidonic acid appeared to be hydroxylated at C13, C20 and, to some extent, at C10, C19 and C7. Fetal liver microsomes mainly formed 13R-HETE, whereas adult human liver microsomes and CYP1A2 mainly formed 13S-HETE. 7,8-Benzoflavone (5 μM) and furafylline (20 μM), two inhibitors of CYP1A2, reduced the bisallylic hydroxylation activity of adult human liver microsomes. Treatment of rats with erythromycin or dexamethasone induced bisallylic hydroxylation of linoleic acid to 11-HODE in liver microsomes by 2- and 10-fold, respectively. The biosynthesis of 11-HODE by microsomes of dexamethasone-treated rats was inhibited by troleandomycin (ED50 = 1 μM) and by polyclonal antibodies against CYP3A1, suggesting that CYP3A1 could catalyze bisallylic hydroxylations in the dexamethasone-treated rat. We conclude from steric analysis of 13-HETE and the effects of CYP inhibitors on adult human liver microsomes that CYP1A2 might contribute to its bisallylic hydroxylation activity.

Liver and renal cortical microsomes and NADPH oxygenate polyunsaturated fatty acids to a large number of metabolites (Capdevila et al., 1995; Oliw, 1994; Oliw et al., 1996). Many of the oxidizing enzymes have been identified as members of the P450 superfamily of heme-thiolate enzymes. Hydroxylations of carbons near or at the ω end and epoxidations of the double bonds by CYPs have attracted the largest attention due to the biological activities of these metabolites of arachidonic acid (Capdevila et al., 1995; McGiff, 1991;Oliw, 1994; Oliw et al., 1996).

The CYP enzymes are important for metabolism of other endogenous compounds like vitamin D, steroids, bile acids, prostaglandins and leukotrienes as well as xenobiotics (Denison and Whitlock, 1995; Nelsonet al., 1996). Some CYP enzymes catalyze more or less regiospecific oxygenations with fatty acids as substrates. The CYP4A subfamily catalyzes ω1 and ω2 hydroxylation of polyunsaturated fatty acids (Capdevila et al., 1995; Oliw, 1994), whereas CYP2E1 and other isozymes have been reported to catalyze ω2 and ω3 hydroxylations of arachidonic acid (Laethem et al., 1993;Rifkind et al., 1995). Some human and rodent epoxygenases have also been identified in the liver and kidney. For example, CYP1A2 and isoforms of CYP2B and CYP2C catalyze epoxygenation of arachidonic acid (Capdevila et al., 1995; Daikh et al., 1994;Oliw, 1994; Zeldin et al., 1995), but it seems likely that many other constitutive enzymes with epoxygenase activity remain to be identified both in liver and in extrahepatic organs.

Liver microsomes can catalyze two additional NADPH-dependent oxygenation reactions: bisallylic hydroxylation and hydroxylation with double bond migration (Oliw et al., 1993). Bisallylic hydroxylation in liver microsomes of phenobarbital-treated rats converts linoleic acid to 11-HODE and arachidonic acid to 7-HETE, 10-HETE and 13-HETE (Brash et al., 1995; Hörnstenet al., 1996; Oliw et al., 1993). These metabolites are acid labile and decompose to cis-transconjugated dienols nonenzymatically (Hamberg et al., 1992). Hydroxylation with double bond migration, which also has been referred to as allylic hydroxylation (Capdevila et al., 1995), leads to enzymatic biosynthesis of cis-trans conjugated hydroxy fatty acids. The mechanism has been investigated in microsomes of phenobarbital-treated rats with linoleic acids stereospecifically deuterated at C11 as substrates (Oliw et al., 1993). Linoleic acid was converted to 9-HODE and 13-HODE with 80% to 82%R stereochemistry by initial hydrogen abstraction at C11 with subsequent double bond migration and oxidation at C9 or C13. The same system oxygenated arachidonic acid to 12-HETE (80% R) in moderate amounts. Human liver microsomes can also form 13-HETE and 12R-HETE (Oliw, 1993). The mechanism of biosynthesis of 12R-HETE by liver microsomes has not been determined, but it seems likely that it occurs by hydroxylation with double bond migration. An alternative route may be present in skin, in which 12R-HETE might be formed by other mechanisms (McGiff, 1991; Oliw, 1994). 12R-HETE is a partial agonist of the leukotriene B4 receptor and it has therefore attracted some biological attention (McGiff, 1991).

The CYP enzymes that catalyze bisallylic hydroxylation and hydroxylation with double bond migration have not been identified. Phenobarbital treatment of rats increased the bisallylic hydroxylation activity of liver microsomes by only a little. Treatment of rats with DEX, which mainly induces the CYP3A subfamily in the rat liver (Pereira and Lechner, 1995), increased the bisallylic hydroxylation activity of liver microsomes on linoleic, arachidonic and eicosapentaenoic acids (Hörnsten et al., 1996). Bisallylic hydroxylation activity could also be increased by treatment of rats with acetone plus starvation, with imidazole and with β-naphthoflavone, indicating that other enzymes than the CYP3A subfamily could have this enzyme activity in the rat.

The CYP enzymes with bisallylic hydroxylation activity can be conveniently identified with the help of recombinant enzymes. Bisallylic hydroxylation activity of human recombinant CYP has not been investigated, but a recent report addresses this question indirectly.Rifkind et al. (1995) found that CYP1A2, CYP2C8 and CYP2C9 converted arachidonic acid to relatively large amounts ofcis-trans conjugated HETEs. In this study, it is likely that bisallylic hydroxy metabolites may have decomposed tocis-trans conjugated HETEs during the acidic extractive isolation. These investigators made the interesting observation that CYP2C9 metabolized arachidonic acid to 12-HETE, but the stereochemistry of 12-HETE was not examined.

The first objective of the present study was to investigate whether a series of recombinant human CYP enzymes could form bisallylic hydroxy metabolites of linoleic and arachidonic acids. Linoleic acid is a convenient substrate for this purpose, whereas arachidonic acid can be oxygenated to a much more complex mixture of metabolites. Biosynthesis of 12-HETE by CYP2C9 and its allelic variant R144C was also investigated. The second objective was to use the results from the screening of the CYP enzymes to study their contribution to the bisallylic hydroxylation activity of adult human liver microsomes. This was done by the aid of enzyme inhibitors and by chiral analysis of the main product, 13-HETE. Thus, we examined the effects of some enzyme-specific inhibitors on bisallylic hydroxylation activity of adult human liver microsomes and compared the chirality of 13-HETE formed by CYP and by liver microsomes. We also found that human fetal liver microsomes catalyzed bisallylic hydroxylation of fatty acids. Finally, the effects of gender, starvation and treatment with ERY and DEX on rat liver bisallylic hydroxylation activity were examined.

Experimental Procedures

Materials.

18:2n-6 (99%), 20:4n-6 (99%), DEX, phenylmethylsulfonyl fluoride, testosterone, 7,8-benzoflavone, ERY and TAO were from Sigma Chemical (St. Louis, MO). [1-14C]18:2n-6, [1-14C]20:4n-6 (55 Ci/mol) and [4-14C]testosterone (56 Ci/mol) were from Amersham (Amersham, UK). The radiolabeled fatty acids were usually diluted with unlabeled fatty acids to a specific activity of 2.3 Ci/mol. 6β-Hydroxytestosterone was from Steraloids (Wilton, NH). Cartridges for extraction (SepPak/C18) were from Waters (Milford, MA). 13S-HODE and 15S-HETE were obtained through biosynthesis (Oliw et al., 1993). 9R,S-HETE, 11R,S-HETE, 12S-HETE and 20-HETE were from Cayman Chemical (Reading, UK). 12R-HETE methyl ester was a gift of Dr. S.-E. Dahlén (Karolinska Institutet, Stockholm, Sweden). 18R-HETE and 19R-HETE were obtained by biosynthesis with mycelia of Gäumannomycis graminis (Oliw, 1989), whereas 17-HETE was a gift from Dr. J. R. Falck (University of Texas Southwestern Medical Center, Dallas, TX). Sulfaphenazole and furafylline were purchased from Ultrafine Chemicals (Manchester, UK). Microsomes of recombinant human P450 expressed in lymphoblastoid cells (CYP1A2, CYP2A6, CYP2B6, CYP2D6 and CYP2E1) and insect cells [CYP3A4, CYP2C8, CYP2C9 (with Arg144 or Cys144) and CYP2C19] were purchased from Gentest (Woburn, MA). Three samples of human livers were obtained from the liver bank of Huddinge Hospital (courtesy of Dr. J. Säwe and Dr. M.-L. Dahl): HL37 was from a 59-year-old woman who died from head trauma after treatment with mannitol, ekvacillin and atropine; HL42 from a 31-year-old man who died after subarachnoidal bleeding after treatment with theophylline and phenytoin; and HL46 was from an adult male with an unknown case history. Two samples of human fetal livers (before week 24 of gestation) were kindly provided by Dr. A. Rane (Akademiska Sjukhuset, Uppsala, Sweden). Polyclonal rabbit antibodies against CYP3A1 were purchased from XenoTech (Kansas City, KS) and were used for inhibition of CYP3A1 as recommended by the supplier.

Incubation with microsomes of recombinant human P450 expressed in insect or lymphoblastoid cells.

From 25 to 50 pmol of CYP3A4, CYP2C8, CYP2C9 or CYP2C19 in 0.5 to 1 ml of incubation buffer (0.05 mM Tris-HCl, pH 7.4, containing 1 mM EDTA, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride and 0.5 mM dithiothreitol) were incubated with 14C-labeled fatty acids (4–5 μM; 56 Ci/mol) and NADPH (1 mM) for 30 min at 37°C. P450 expressed in lymphoblastoid cells were incubated for 2 hr at 37°C (≈30–65 pmol of CYP1A2 and CYP2A6, 45–90 pmol of CYP2E1, 65–130 pmol of CYP2D6 and 50–100 pmol of CYP2B6) with 15 to 20 μM of substrate. Microsomes of insect and lymphoblastoid cells without expressed P450 were incubated in the same way. Incubations were terminated by four volumes of ethanol. An internal standard (13-HODE or 15-HETE) was added to check for recovery on HPLC and used for estimation of biosynthesis of metabolites during the incubation time period and for comparison between enzymes (tables 1 and2). The standards also verified the retention times on HPLC.

View this table:
Table 1

Metabolism of linoleic acid by recombinant human CYP and by adult and fetal human liver microsomes to epoxides/diols, 11-HODE, 17-HODE (ω2) and 18-HODE (ω1)

View this table:
Table 2

Metabolism of arachidonic acid by adult and fetal human liver microsomes and recombinant human CYP to epoxides/diols, 20-HETE, 19-HETE and other HETEs

Incubation with human and rat liver microsomes.

Human liver microsomes were prepared by differential centrifugation as described and stored at −80°C until use (Oliw, 1993). Rat liver microsomes were prepared separately in the same way from each animal. Fischer 344 rats (170–190 g; Mollegaard, Skensved, Denmark) were treated with DEX (150 mg/kg for 4 days; three males, one female), with ERY (734 mg/kg p.o. for 5 days, n = 3) or with solvent (1% Tween-20; three males, one female) as described previously (Daujat et al., 1991). Three male rats were also starved for 48 hr.

Microsomes were suspended in 1 to 2 ml of incubation buffer as described above to a protein concentration of ≈1 mg/ml and incubated with 1.0 mM NADPH and the 14C-labeled fatty acids (usually 0.1 mM, 2.3 Ci/mmol) for 30 min at 37°C with constant shaking. Incubations were terminated as above. The effects of mechanism-based inhibitors (furafylline and TAO; both dissolved in methanol) were assessed after preincubation with the drug and NADPH for 15 min at 37°C. [14C]18:2 or [14C]20:4 was then added to the incubation, which was terminated after 30 min as described above. Sulfaphenazole and 7,8-benzoflavone were added directly to the incubations without preincubations. Control incubations and incubations with drugs were performed in duplicate.

Purification of metabolites.

After extractive isolation without acidification on a cartridge with octadecasilane silica (SepPak/C18), the arachidonic acid metabolites were first separated by RP-HPLC into three groups of metabolites: triols and diols, HETEs and epoxides. HETEs typically eluted between 20 and 30 min. These fractions with HETEs were then pooled and analyzed by SP-HPLC. 13-HETE and 19-HETE have similar retention times on RP-HPLC (Brash et al., 1995), and in some experiments, unlabeled 19R-HETE (25–50 μg; monitored by UV at 207 nm) was added to localize the 13-HETE metabolite in the chromatogram. Unlabeled 12-HETE was added to incubations with CYP2C9 to facilitate identification of radiolabeled 12R-HETE by RP-HPLC, SP-HPLC and chiral HPLC. The metabolites of linoleic acid were extracted as above and separated by RP-HPLC. The 11-HODE peak was further analyzed by SP-HPLC and GC-MS in some experiments.

HPLC.

The equipment for HPLC has been described (Hörnsten et al., 1996). In short, the columns contained octadecasilane silica (5-μm, 250 × 4.6 mm) for RP-HPLC and silica (5-μm, 250 × 4.6 mm) for SP-HPLC. The RP-HPLC column was eluted with methanol/water/acetic acid (75:25:0.01) at 1 ml/min, and the SP-HPLC column was eluted with hexane/isopropanol/acetic acid (98:2:0.1) at 0.5 to 1 ml/min. The elution order of HETEs on SP-HPLC was 12-HETE, 15-HETE, 11-HETE, 13-HETE, 18-HETE, 10-HETE, 19-HETE, 7-HETE and 20-HETE.

Three different columns were used for chiral HPLC: silica with (R)-(−)-N-3,5-dinitrobenzoyl-α-phenylglycine (ionically bonded; 5 μm, 250 × 4.6 mm; Oliw et al., 1993) and Chiralcel OB-H for analysis of 12-HETE and Chiralcel OD-H (5 μm, 250 × 4.6 mm) for analysis of 13-HETE (Brash et al., 1995). These chiral columns were eluted with 0.5%, 2% and 2% isopropanol in hexane, respectively, at 0.5 to 1.0 ml/min. The HPLC columns were connected to a photo diode array detector (Waters 991) and to a radioactivity detector for HPLC (Radiomatic Flo-one 150TR, Packard) using Ultima Flo AP (Packard) as scintillator. The internal standard (13-HODE, 15-HETE) was monitored by UV absorption at 235 nm during HPLC. In addition, large amounts of the bisallylic hydroxy metabolites could be monitored by their end absorption at 205 to 220 nm (Brash et al., 1995).

Derivatizations and GC-MS analysis.

Trimethylsilyl ethers were prepared by treatment with bis(trimethylsilyl)trifluoroacetamide and pyridine and methyl esters with diazomethane (Oliw et al., 1993). A capillary GC (Varian 3100) with a nonpolar column (30-m; DB-5, J&W Scientific; film, 0.25 μm; diameter, 0.25 mm; carrier gas He, 15 p.s.i.) and an ion trap mass spectrometer (ITS40, Finnigan MAT) were used as described (Oliw et al., 1993). C-values were determined by the retention times of fatty acid methyl esters (18:0, 20:0, 22:0 and 24:0). Steric analysis of 11-HODE was performed after partial hydrogenation and ozonolysis of the (−)-menthoxycarbonyl derivative as described previously (Hamberget al., 1992).

Other analyses.

Protein was determined as described byBradford (1976) using γ-globulin as a standard. Testosterone 6β-hydroxylase activity was assayed according to directions of the supplier of [14C]testosterone (Amersham) using TLC on silica plates (20 × 20 cm; Whatman LK6D, Maidsone, UK; eluent CH2Cl2/acetone, 4:1) for separation. Radioactivity of the TLC plates was determined by a TLC scanner (Berthold Dünnshichtsscanner II; Kebo, Stockholm, Sweden). The Rf values were 0.5 and 0.25 for testosterone and 6β-hydroxytestosterone, respectively. O-Dealkylation of ethoxyresorufin and 4′-hydroxylation of diclofenac sodium by human liver microsomes were assayed as described previously (Burke et al., 1985; Leemann et al., 1992).

Results

Recombinant human CYP and linoleic acid.

RP-HPLC of metabolites of [14C]18:2n-6, which were formed by NADPH and CYP1A2 (60 pmol, 2 hr) expressed in lymphoblastoid cells and by CYP2C8 (25 pmol, 0.5 hr), CYP2C9 (50 pmol, 0.5 hr) and CYP3A4 (25 pmol, 0.5 hr) expressed in insect cells, are shown in figure1, A–D. All four enzymes formed a prominent metabolite with an elution time of ≈17 to 18 min. This metabolite had the same retention time as [14C]11-HODE formed by liver microsomes of DEX-treated rats (Hörnsten et al., 1996). CYP1A2 and CYP3A4 formed this metabolite, 11-HODE, as a major product. A fifth enzyme, CYP2C19, also formed some 11-HODE (table 1).

Figure 1
Figure 1

RP-HPLC of metabolites formed from [14C]linoleic acid by CYP1A2 (A), CYP2C8 (B), CYP2C9 (C) and CYP3A4 (D). •, Peak in the chromatograms had the same elution time as [14C]11-HODE and was totally absent in control insect cells and control lymphoblastoid cells. The other products were not characterized, but diols, 17-HODE, 18-HODE and monoepoxides of 18:2n-6 eluted after ≈13, ≈21, ≈23 and ≈30 to 34 min, respectively.

In addition to 11-HODE, 14C-labeled compounds eluted with the same retention time as 17-HODE (≈21 min), 18-HODE (≈23 min) and monoepoxides of 18:2n-6 (≈32 min) in all chromatograms (fig. 1) and in experiments with CYP2C19. CYP1A2 appeared to form larger amounts of 17-HODE (fig. 1A) than the other enzymes. Epoxides were partly converted to diols, which eluted after 13 min, by microsomes of insect cells (e.g., fig. 1, C and D) but not by microsomes of lymphoblastoid cells (fig. 1A). Finally, CYP2C9 and its allelic variant R144C metabolized 18:2n-6 to the same profile of metabolites.

The total biosynthesis of metabolites of 18:2n-6 by the enzymes with bisallylic hydroxylation activity and the relative formation of major metabolites are summarized in table 1. The main products formed by all the enzymes were epoxides/diols, ω1 and ω2 hydroxy metabolites, which is in agreement with previous studies (Oliw, 1994). The bisallylic metabolite 11-HODE is nevertheless a relatively prominent metabolite, particularly of CYP1A2 and CYP3A4.

Control microsomes of insect and lymphoblastoid cells metabolized [14C]18:2n-6 insignificantly and no radioactivity eluted between 15 and 20 min (i.e., in the part of the chromatogram in which [14C]11-HODE eluted). CYP2A6, CYP2B6, CYP2D6 and CYP2E1 did not form significant amount of [14C]11-HODE as judged from RP-HPLC.

Attempts to obtain 11-HODE in sufficient amounts for GC-MS analysis by using larger amounts of recombinant enzyme and higher concentrations of substrate were unsuccessful. Incubation with 0.1 mM 18:2n-6 (or 20:4n-6) appeared to lead to enzyme inactivation, in agreement with a previous report (Imaoka et al., 1992).

Recombinant human CYP and arachidonic acid. CYP1A2 metabolized [14C]20:4n-6 to many polar products as judged from RP-HPLC (fig. 2A). The main metabolites were HETEs and epoxides with little hydrolysis to diols (not shown). When the HETEs were analyzed by SP-HPLC, several major metabolites were separated, as shown in figure 2B. The major metabolites had the same retention times as 13-HETE, 19-HETE, 7-HETE and 10-HETE, with only little radioactivity eluting at the retention time of 20-HETE. We noted a backside shoulder on the 13-HETE peak. It has been reported that 16-HETE and 17-HETE have a slightly longer retention time than 13-HETE (see Brash et al., 1995), and these metabolites might contribute (Falck et al., 1990). 18-HETE also elutes after 13-HETE.

Figure 2
Figure 2

HPLC of metabolites formed from [14C]arachidonic acid by human recombinant CYP1A2 and CYP3A4. A, RP-HPLC of metabolites formed from CYP1A2. The material, which eluted after 20 to 29 min, was combined, designated HETEs and analyzed by SP-HPLC. B, SP-HPLC of HETEs from A. C, SP-HPLC of HETEs formed by CYP3A4. D, SP-HPLC of HETEs formed by liver microsomes of DEX-treated rats. These microsomes generate a mixture of 13-HETE, 10-HETE, 7-HETE as well as 19-HETE and 20-HETE, which have been characterized previously.

CYP3A4 formed 13-HETE as the major monohydroxy metabolite, followed by 10-HETE and 7-HETE as judged from SP-HPLC (fig. 2C). Biosynthesis of the ω1 and ω2 hydroxy metabolites was insignificant. As discussed above, we could not obtain sufficient material for GC-MS analysis by recombinant enzymes. To correct for day-to-day variations in retention times of SP-HPLC, we used UV monitoring of internal standards (13-HODE, 15-HETE). We also analyzed pooled monohydroxy metabolites of [14C]20:4n-6 formed by rat liver microsomes of DEX treated rats for comparison (fig. 2D). The major metabolites of the peaks in the chromatogram (marked 7-, 10-, 13-, 19- and 20-HETE in fig.2D) were previously identified by GC-MS (Hörnsten et al., 1996).

CYP2C9 formed two major peaks on SP-HPLC (fig.3A), which eluted after ≈7 and ≈10 min, respectively, as well as many minor metabolites. The first metabolite had the same retention time as 12-HETE on SP-HPLC (on coinjection; fig. 3B), and it eluted mainly with the 12R-HETE stereoisomer on chiral HPLC (fig. 3C). The second14C-labeled metabolite had the same retention time as 13-HETE.2 The two metabolites were therefore tentatively identified as 12R-HETE and 13-HETE. In addition, radiolabeled material with the same elution time as 15-HETE (directly after 12-HETE), 11-HETE (directly before 13-HETE), 10-HETE (after 13-HETE) and 19-HETE eluted during SP-HPLC, but there was negligible biosynthesis of 7-HETE and 20-HETE. The allelic variant R144C of CYP2C9 yielded similar results. CYP2C8 converted [14C]20:4n-6 to main hydroxy metabolites with the same retention times as 13-HETE, 15-HETE, 11-HETE, 10-HETE and 19-HETE on SP-HPLC (data not shown).

Figure 3
Figure 3

Analysis of HETEs formed by human recombinant CYP2C9 from [14C]arachidonic acid. A, SP-HPLC of HETEs. The peaks marked 12-HETE, 13-HETE and 19-HETE had the same retention times as these compounds (see text for details). B, SP-HPLC with UV detection and radioactivity monitoring of the first eluting14C-labeled metabolite (marked 12-HETE in A) after the addition of authentic 12-HETE. C, Chiral HPLC of the first eluting radiolabeled peak in A (12-HETE) with added 12S-HETE and 12R-HETE. The14C-labeled metabolite eluted mainly with the 12R-HETE stereoisomer.

Steric analysis of 12-HETE formed by CYP2C9 and by its allelic form R144C (with base-line separation of the antipodes on Chiralcel OB-H) showed that it consisted of >95% 12R-HETE.

CYP2C19 formed 19-HETE as the main and prominent metabolite with relatively little biosynthesis of bisallylic hydroxy metabolites and other HETEs. This enzyme also formed epoxides/diols as major metabolites.

The percent distribution of the arachidonic acid metabolites into epoxides/diols and HETEs by the CYP enzymes with bisallylic hydroxylation activity is summarized in table2 together with an estimate of the amounts of 20-HETE, 19-HETE and other HETEs (including the bisallylic hydroxy metabolites). Control microsomes without expressed CYP did not significantly metabolize arachidonic acid.

Bisallylic hydroxylation of linoleic and arachidonic acids by fetal and adult human liver microsomes. Fetal liver microsomes converted [14C]18:2n-6 to [14C]11-HODE as judged from RP-HPLC (fig.4A). This metabolite was also identified by GC-MS. The product was formed in similar amounts as 18-HODE and epoxides/diols (table 1). Two different samples of human fetal liver yielded similar results.

Figure 4
Figure 4

RP-HPLC analysis of metabolites of [14C]linoleic acid formed by human liver microsomes and NADPH. A, Fetal liver microsomes. B, Adult human liver microsomes. •, Peak marked in the chromatograms had the same elution time as [14C]11-HODE.

[14C]20:4n-6 was converted by fetal liver microsomes to 20-HETE and 13-HETE as major monohydroxy metabolites by two samples of fetal liver microsomes (table 2). Radiolabeled material with the retention times of 10-HETE, 7-HETE and 19-HETE was also noted on SP-HPLC.

Adult human liver microsomes from three patients (HL37, HL42 and HL46) were also investigated. 18:2n-6 was mainly converted to 18-HODE and diols, and only small amounts of 11-HODE were formed by the three samples (HL37; fig. 4B). However, in the presence of 7,8-benzoflavone (100 μM), the amounts of 11-HODE appeared to increase 2- to 3-fold in the three liver samples (data not shown). The stimulatory effect of 7,8-benzoflavone appeared to be even more striking when [14C]20:4n-6 was used as a substrate. Figure5, A and B, shows the monohydroxy metabolites of [14C]20:4n-6 formed by human liver microsomes (HL37) with and without 7,8-benzoflavone (0.1 mM) present, respectively. 7,8-Benzoflavone appeared to markedly increase the biosynthesis of 7-HETE and decrease biosynthesis of 20-HETE in comparison with the control incubations. HL42 and HL46 yielded similar results. 7,8-Benzoflavone is a selective inhibitor of CYP1A2 at low concentrations (1–10 μM), whereas high concentrations (100 μM) inhibit many different CYP enzymes but can augment the activity of CYP3A4 (Newton et al., 1995; Ono et al., 1996;Schwab et al., 1988; Yun et al., 1992). A low concentration of 7,8-benzoflavone (5 μM) was found to reduced the biosynthesis of 11-HODE (HL42 and HL46), as discussed below.

Figure 5
Figure 5

RP-HPLC analysis of metabolites of [14C]arachidonic acid formed by human liver microsomes. A, From an incubation of human liver microsomes (HL37) and NADPH in the presence of 7,8-benzoflavone (100 μM). B, Incubation of the same microsomes and NADPH but without 7,8-benzoflavone. αNF designates 7,8-benzoflavone (α-naphthoflavone).

Effect of CYP inhibitors on adult human liver microsomes.

The screening of CYP enzymes indicated that CYP3A4, the CYP2C subfamily and CYP1A2 might contribute to the bisallylic hydroxylation activity of human microsomes. Specific inhibitors of these enzymes were studied.

TAO (10–50 μM) did not affect the bisallylic hydroxylation of three different adult human liver microsomes. TAO is a mechanism-based inhibitor of the CYP3A subfamily, including CYP3A4 (Newton et al., 1995; Ono et al., 1996). To confirm that concentration of TAO was sufficient, we confirmed that 10 μM TAO reduced 6β-hydroxylation of testosterone in these human microsomes by >90% (see Newton et al., 1995). In addition, TAO (1–10 μM) inhibited the biosynthesis of 11-HODE from 18:2n-6 by liver microsomes of DEX-treated rats (see below).

We also investigated the effects of sulfaphenazole (50 μM), an inhibitor of the CYP2C subfamily (except CYP2C19), and two inhibitors of CYP1A2, furafylline (20 μM), a mechanism-based inhibitor and 7,8-benzoflavone (Crespi et al., 1997; Newton et al., 1995; Ono et al., 1996), on the bisallylic hydroxylation of 18:2n-6 by three different samples of adult human liver microsomes (HL37, HL42, HL46).

Sulfaphenazole did not inhibit the bisallylic hydroxylation of linoleic and arachidonic acids, but there appeared to be some reduction in the biosynthesis of epoxides/diols activity, in agreement with studies byRifkind et al. (1995). We confirmed that drug concentration was sufficient. Sulfaphenazole reduced by >90% the 4′-hydroxylation of diclofenac (HL42, HL46).

Furafylline (20 μM; three livers) and 7,8-benzoflavone (5 μM: HL42, HL46) reduced the bisallylic hydroxylation activity on 18:n-6 by 60% and 40% to 50%, respectively. At this concentration both drugs inhibited the O-dealkylation of ethoxyresorufin by >90% (HL42, HL46).

Effect of CYP inhibitors on fetal liver microsomes.

Fetal liver microsomes contain CYP3A7. TAO is also an inhibitor of CYP3A7 (Hashimoto et al., 1995), but TAO (10 μM) did not reduce the biosynthesis of 11-HODE by fetal liver microsomes. Furafylline (20 μM) did not inhibit the biosynthesis of 11-HODE by fetal liver microsomes.

Steric analysis of 13-HETE.

The stereochemistry of 13-HETE formed by human liver microsomes and some recombinant CYP was determined by chiral HPLC (Chiralcel OD-H) as described previously (Brash et al., 1995). As a standard, we used 13-HETE formed by liver microsomes of phenobarbital-treated rats, which was found to contain 60% 13R-HETE (see Brash et al., 1995).

Human and fetal liver microsomes appeared to form 13-HETE with different chirality. Two samples of fetal liver microsomes mainly formed 13R-HETE (75–80%), whereas three samples of adult human liver mainly formed 13S-HETE (73–90%) as judged from chiral HPLC (fig.6, A, B and D). The results indicated that adult and fetal liver microsomes form 13-HETE by different enzymes.

Figure 6
Figure 6

Steric analysis of 13-HETE methyl ester obtained from incubations with fetal and adult human liver microsomes using chiral HPLC. A, 13-HETE obtained from fetal liver microsomes. B, 13-HETE from adult human liver microsomes (HL46). C, 13-HETE from adult human liver microsomes (HL42) incubated with 100 μM 7,8-benzoflavone. D, 13-HETE from liver microsomes (HL42) incubated without 7,8-benzoflavone. Before chiral HPLC, 13-HETE was purified by RP- and SP-HPLC.

In the presence of 7,8-benzoflavone (100 μM), adult human livers formed 13-HETE with much less stereospecificity (fig. 6, C and D). As discussed above, 7,8-benzoflavone (100 μM) can augment the enzyme activity of CYP3A4. We found that CYP3A4 formed virtually racemic 13-HETE on Chiralcel OD-H. It therefore seems likely that CYP3A4 may contribute to the increase in bisallylic hydroxylation activity of human liver microsomes in the presence of stimulatory concentrations of 7,8-benzoflavone.

Steric analysis of 13-HETE from CYP1A2 showed that it contained 75% of 13S-HETE and 25% 13R-HETE. CYP1A2 and human liver microsomes thus mainly formed the 13S-antipode of 13-HETE (75% and 73–90%, respectively). In some analysis of 13-HETE methyl ester (CYP1A2, human liver microsomes) on chiral HPLC, a peak of radioactivity eluted with the same retention time as authentic 17-HETE methyl ester, ≈4 min before 13S-HETE methyl ester. Analysis of 13-HETE from incubations with CYP3A4 and from microsomes of DEX-treated rats did not show significant amounts of this material.

Liver microsomes of Fischer rats.

11-HODE was formed as a minor metabolite of 18:2n-6 by liver microsomes of untreated rats. Starvation for 48 hr did not appear to increase its biosynthesis (n = 3), whereas treatment with starvation and acetone has been found to do so previously (Hörnsten et al., 1996). ERY treatment appeared to induce the biosynthesis of 11-HODE ≈2-fold, and DEX treatment induced biosynthesis by ≈10-fold. We confirmed that both treatments strongly (5–20-fold) induced the 6β-hydroxylation of testosterone in liver. We also found that DEX treatment increased bisallylic hydroxylation activity in liver microsomes of both sexes. As expected, there appeared to be an increased bisallylic hydroxylation of 20:4n-6 after treatment with DEX: ≈2-fold for 13-HETE and 10-HETE in some experiments and ≈4-fold for 7-HETE (data not shown). 13-HETE prepared by liver microsomes of DEX-treated rats was found to be racemic by chiral HPLC.

DEX and ERY are known to induce the CYP3A subfamily, particularly CYP3A1 and closely related enzymes, in the rat liver (Daujat et al., 1991). CYP3A1 was therefore an obvious candidate with bisallylic hydroxylation activity. This possibility was assessed by TAO, an inhibitor of the CYP3A subfamily (Newton et al., 1995; Ono et al., 1996), and by inhibitory antibodies against CYP3A1. TAO dose-dependently reduced the biosynthesis of 11-HODE with an ED50 of 1 μM (fig.7). TAO caused 85% inhibition at 10 μM. TAO (10 μM) also appeared to reduce the bisallylic hydroxylation of 20:4n-6 to 13-HETE and 7-HETE by >50%. Finally, polyclonal antibodies against CYP3A1 (1 mg/ml) were found to inhibit almost completely the bisallylic hydroxylation of [14C]18:2n-6 by liver microsomes of DEX-treated rats, whereas the control incubation, which contained IgG (1 mg/ml) instead, formed [14C]11-HODE (data not shown).

Figure 7
Figure 7

Effect of troleandomycin on the biosynthesis of [14C]11-HODE from [14C]linoleic acid by liver microsomes of DEX-treated rats.

GC-MS analysis and steric analysis of 11-HODE.

11-HODE was identified from an incubation with fetal liver microsomes with 18:2n-6 and NADPH. The C-value and the mass spectrum were as reported previously (Hamberg et al., 1992). The stereochemistry of 11-HODE formed by liver microsomes of DEX treated rats was found to be nearly racemic (58% R). 11-HODE is also formed by liver microsomes of phenobarbital-treated rats, and in this case the result was almost identical, 59% 11R-HODE (Oliw et al., 1993).

Discussion

The present study focuses on bisallylic hydroxylation and hydroxylation with double bond migration of polyunsaturated fatty acids. Polyunsaturated fatty acids are also metabolized to epoxides and to ω1 and ω2 hydroxy metabolites as major products (Capdevilaet al., 1995; Oliw, 1994; Oliw et al., 1996). These reactions can be catalyzed by many different CYP enzymes and have been studied extensively, whereas the present work is the first systematic study of human recombinant CYP with bisallylic hydroxylase activity.

Human recombinant enzymes.

We report three new findings. Five of 10 human recombinant CYP, viz., CYP1A2, CYP3A4, CYP2C8, CYP2C19 and two isoforms of CYP2C9 (Arg144 and Cys144), were found to metabolize 18:2n-6 to 11-HODE and 20:4n-6 to 13-HETE. CYP1A2 and CYP3A4 also hydroxylated the two other bisallylic carbons of 20:4n-6, although 13-HETE was clearly the main bisallylic hydroxy metabolite. CYP2C9 and CYP2C8 yielded a different pattern. The former also oxidized 20:4n-6 to 12R-HETE as a main metabolite and to small amounts of 15-HETE, 11-HETE and 10-HETE. The latter formed 13-HETE along with 15-HETE, 11-HETE and small amounts of 10-HETE. CYP1A2 and CYP3A4 can thus catalyze bisallylic hydroxylations, whereas CYP2C8 and CYP2C9 also can formcis-trans conjugated HETEs, presumably by hydroxylation with double bond migration. Some of the main hydroxy metabolites formed by these five enzymes are summarized in figure8.

Figure 8
Figure 8

Overview of bisallylic hydroxylations and hydroxylation with double bond migration. A, Bisallylic hydroxylation of linoleic acid to 11-HODE (1). B, Bisallylic hydroxylation of arachidonic acid to 7-HETE (2), 10-HETE (3) and 13-HETE (4). C, Hydroxylation with double bond migration of arachidonic acid to 12R-HETE (5) and bisallylic hydroxylation to 13-HETE (6). In addition, all five recombinant enzymes also formed epoxides/diols, and many of them formed hydroxylated carbons of the ω side chain.

Our second finding, which is based on specific inhibitors and steric analysis of 13-HETE, is that CYP1A2 could contribute to the bisallylic hydroxylation activity of adult human liver microsomes. CYP1A2, CYP3A4 and the CYP2C subfamily together make up ≈60% of the CYP enzymes of human liver, but there are large interindividual variations (Guengerich, 1995). In view of the bisallylic hydroxylation activity of the recombinant enzymes discussed above, we expected that specific inhibitors of CYP1A2, the CYP2C subfamily and CYP3A4 would reduce the bisallylic hydroxylation activity of human liver microsomes. Only inhibitors of CYP1A2 (furafylline, 7,8-benzoflavone) appeared to do so, whereas inhibitors of the CYP3A subfamily (TAO) and the CYP2C subfamily (sulfaphenazole) were without effects.

Furafylline and low concentrations of 7,8-benzoflavone are known to inhibit CYP1A2 with selectivity (Newton et al., 1995; Onoet al., 1996). Both drugs reduced the bisallylic hydroxylation of 18:2n-6 by 60% and 40%, respectively. In addition, both CYP1A2 and adult human liver microsomes mainly formed the 13S stereoisomer of 13-HETE. The selective inhibitors and the steric analysis suggested that CYP1A2 could contribute to the bisallylic hydroxylation activity of adult human liver microsomes in vitro, but other CYP enzymes might also contribute.

Our third finding is that fetal liver, microsomes had a prominent bisallylic hydroxylation activity on 18:2n-6 and 20:4n-6 in comparison with the epoxygenation and hydroxylation of these fatty acids. Interestingly, the chirality of 13-HETE formed by adult and fetal liver microsomes differed. 13-HETE from fetal liver contained >70% of theR stereoisomer, and that from adult liver contained >70% of the S stereoisomer. This finding suggested that fetal and adult liver microsomes formed 13-HETE by different enzymes. Neither TAO nor furafylline reduced the bisallylic hydroxylation activity of fetal liver microsomes.

The lack of effect of TAO on the bisallylic hydroxylation activity of adult and fetal human microsomes was unexpected. CYP3A4 and CYP3A7 are quantitatively major enzymes in adult and fetal human liver, respectively (Guengerich, 1995). We also confirmed that our human adult microsomes catalyzed a typical CYP3A4 reaction, the 6β-hydroxylation of testosterone. The difference between human liver microsomes and recombinant CYP3A4 could be related to differences in coupling between cytochrome P450 reductase and CYP3A4. The recombinant CYP3A4 was coexpressed with NADPH-P450 reductase. The enzymatic activity of CYP3A4 in microsomes can be augmented by 0.1 mM 7,8-benzoflavone (Berthouet al., 1994; Schwab et al., 1988; Yun et al., 1992). The mechanism is unknown, but the drug may facilitate electron flow from the reductase to the monooxygenase. We observed that 7,8-benzoflavone (0.1 mM) could increase the bisallylic hydroxylation activity of adult human liver microsomes. 13-HETE, which was formed in the presence of 7,8-benzoflavone, contained more 13R-HETE and was thus formed with less stereospecificity. CYP3A4 biosynthesized racemic 13-HETE. It therefore seems likely that 7,8-benzoflavone stimulated the bisallylic hydroxylation activity of CYP3A4 in the microsomes.

Rat liver enzymes.

We extended our studies on induction of bisallylic hydroxylation activity of rat liver microsomes (Hörnsten et al., 1996). Treatment with two different inducers of CYP3A1, DEX and ERY, increased the bisallylic hydroxylation activity in rats of both sexes. Furthermore, TAO and polyclonal antibodies against CYP3A1 strongly inhibited this enzymatic reaction. It therefore seems likely that the principal bisallylic hydroxylation activity of liver microsomes of rats treated with DEX and ERY is associated with CYP3A1 or any of its closely related isozymes (Kirita and Matsubara, 1993; Komori and Oda, 1994; Pereira and Lechner, 1995). Phenobarbital treatment can also induce CYP3A1 (Larsen and Jefcoate, 1995). This may contribute to the fact that liver microsomes of DEX and phenobarbital-treated rats formed 11-HODE with the same chirality (58–59% 11R-HETE).

Hydroxylation mechanisms.

Bisallylic hydroxy metabolites are likely formed, in analogy with other hydroxylations, by hydrogen abstraction by the heme ferryl oxygen and “oxygen rebound” (Ortiz de Montellano, 1995). Oxygen is inserted with retention of configuration during biosynthesis of 11-HODE (Oliw et al., 1993). The bisallylic carbon must thus be positioned near the active center of the enzyme. CYP1A2 and CYP3A4 apparently allowed all three bisallylic carbons of 20:4n-6 to interact with the heme iron. CYP2C9 and CYP2C8 differed; they formed 13-HETE as a main hydroxy metabolite, small amounts of 10-HETE and relatively large amounts of certaincis-trans conjugated HETEs (CYP2C9, mainly 12R-HETE; CYP2C8, mainly 11-HETE and 15-HETE). Both enzymes can abstract a hydrogen from C13 and C10 and insert oxygen at these carbons. It seems likely that the double bond may also migrate. After hydrogen abstraction at C13 by CYP2C8, oxygen could be inserted at C11 and C15, yielding 11-HETE and 15-HETE. After hydrogen abstraction at C10 by CYP2C9, oxygen could be inserted at C12, yielding 12R-HETE. Linoleic acid can undergo similar transformations to 11R,S-HODE, 9R-HODE and 13R-HODE by rat liver microsomes (Oliw et al., 1993).

To abstract a hydrogen from a bisallylic carbon requires less energy than from other carbons of the fatty acid (Porter et al., 1995). Alternatively, the reactive ferryl oxygen may to oxidize the double bonds (epoxidation) adjacent to the bisallylic carbon. CYP2C9, for example, formed 14(15)epoxyeicosatrienoic acid as the main metabolite of 20:4n-6. All five CYP enzymes, which catalyzed bisallylic hydroxylations, were also found to epoxidize double bonds. It therefore seems likely that epoxidation and bisallylic hydroxylations could be connected. However, CYP102 of Bacillus megaterium has been reported to have arachidonate ω6 epoxygenase activity without formation of bisallylic or cis-trans conjugated HETEs (Capdevila et al., 1996). All epoxygenases therefore may not catalyze bisallylic hydroxylations.

12R-HETE and 15R-HETE.

CYP2C9 synthesized relatively large amounts of 12-HETE and mainly the 12R antipode. 12R-HETE is also a well known metabolite formed by human and rat liver microsomes (McGiff, 1991). The enantiomeric purity is ≈80% when extracted at acidic pH and >90% at neutral pH. The chirality of 12-HETE suggests that CYP2C9 may contribute to biosynthesis of 12R-HETE by adult human liver microsomes. CYP2C9 could be useful as a model enzyme for studying this reaction in detail. CYP2C9 and its allelic form R144C can hydroxylate a large number of drugs (e.g., tolbutamide, phenytoin, diclofenac sodium and many other nonsteroidal anti-inflammatory drugs;Goldstein and de Morais, 1994), but none of these substrates seems to be subject to hydroxylation with double bond migration.

Adult human liver microsomes also form 15-HETE, which consists predominantly of the 15R antipode (Oliw, 1993). The present work demonstrated that adult human liver microsomes mainly formed 13S-HETE. This is an interesting analogy with oxygenation of 18:2n-6 by rat liver microsomes. Biosynthesis of 11S-HODE and 13R-HODE from stereospecifically deuterated 18:2n-6 occurred by abstraction of thepro-S hydrogen at C11 of 18:2n-6 and suprafacial oxygen insertion (Oliw et al., 1993). From the biosynthesis of 12R-HETE and 10-HETE by CYP2C9, it clearly will be of interest to determine whether this occurs with formation of the Santipode of 10-HETE.

Biological significance of CYP metabolites.

A biological role of epoxides of 20:4n-6, 20-HETE and 19-HETE has been implicated in different organ systems, hormonal signaling and pathophysiological processes (Capdevila et al., 1995; Oliw et al., 1996; Rahman et al., 1997). Cell wall receptors have not yet been identified for these metabolites, but ion channels could be targets for their actions (Li and Campbell, 1997). As is the case with leukotrienes, prostaglandins and other eicosanoids, stereochemical features will determine potency and biological activity. The bisallylic hydroxy metabolites of 20:4n-6 have not yet been investigated for biological potency. Our results demonstrate that 13-HETE is the main bisallylic hydroxy metabolite, which can be formed with different stereoselectivity by human and fetal liver microsomes. The Rand S antipodes of this molecule could be worthy of future biological studies.

Summary.

Adult human liver microsomes, CYP1A2, CYP3A4, CYP2C8, CYP2C9, CYP2C19 and fetal liver microsomes catalyzed bisallylic hydroxylations of 18:2n-6 and 20:4n-6. Effects of specific CYP inhibitors and steric analysis of 13-HETE formed by human liver microsomes, CYP1A2 and CYP3A4 suggested that CYP1A2 could contribute to the bisallylic hydroxylation activity of adult human liver microsomesin vitro, whereas CYP3A4 seemed to be of minor importance. Human fetal liver microsomes possessed a relatively prominent bisallylic hydroxylation activity, which seemed to be unrelated to CYP1A2 and CYP3A7. The fetal CYP enzymes with this catalytic activity should be determined.

Acknowledgments

We thank Dr. M. Hamberg for steric analysis of 11-HODE and Dr. T. Luthman for providing a Chiralcel OD-H column.

Footnotes

  • Send reprint requests to: Dr. Ernst H Oliw, Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala Biomedical Center, Uppsala University, P.O. Box 591, S-751 24 Uppsala, Sweden.

  • ↵1 This work was supported by the Swedish Medical Research Council (Grant 06523). K.V. is supported by the Swedish Institute.

  • ↵2 This was identified by liquid chromatography-mass spectrometry (J. Ericsson, J. Bylund, C. Su and E. H. Oliw, unpublished observation).

  • Abbreviations:
    CYP
    cytochrome P450
    DEX
    dexamethasone
    ERY
    erythromycin
    GC-MS
    gas chromatography-mass spectrometry
    HETE
    hydroxyeicosatetraenoic acid
    HODE
    hydroxyoctadecadienoic acid
    HPLC
    high-performance liquid chromatography
    P450
    cytochrome P450
    RP
    reverse phase
    SP
    straight phase
    TAO
    troleandomycin
    TLC
    thin-layer chromatography
    • Received April 22, 1997.
    • Accepted September 8, 1997.

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OtherPROSTAGLANDINS, LEUKOTRIENES AND OTHER EICOSANOIDS

Cytochromes P450 with Bisallylic Hydroxylation Activity on Arachidonic and Linoleic Acids Studied with Human Recombinant Enzymes and with Human and Rat Liver Microsomes

Johan Bylund, Tina Kunz, Karin Valmsen and Ernst H. Oliw
Journal of Pharmacology and Experimental Therapeutics January 1, 1998, 284 (1) 51-60;
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