Glucuronidation of arachidonic and linoleic acid metabolites by human UDP-glucuronosyltransferases

Arachidonic acids (AA) and linoleic acids (LAs) are metabolized, in several tissues, to hydroxylated metabolites that are important mediators of many physiological and pathophysiological processes. The conjugation of leukotriene B 4 (LTB 4 ), 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, 15-HETE, and 13-hydroxyoctadecadienoic acid (HODE) by the human UDP-glucuronosyltransferase (UGT) enzymes was investigated. All substrates tested were efﬁciently conjugated by human liver microsomes to polar derivatives containing the glucuronyl moiety as assessed by mass spectrometry. The screening analyses with stably expressed UGT enzymes in HK293 showed that glucuronidation of LTB 4 was observed with UGT1A1, UGT1A3, UGT1A8, and UGT2B7, whereas UGT1A1, UGT1A3, UGT1A4, and UGT1A9 also conjugated most of the HETEs and 13-HODE. LA and AA metabolites also appear to be good substrates for the UGT2B subfamily members, especially for UGT2B4 and UGT2B7 that conjugate all HETE and 13-HODE. Inter-estingly, UGT2B10 and UGT2B11,

Linoleic acid (LA), an 18-carbon fatty acid, is an abundant polyunsaturated fatty acid (PUFA) in phospholipids, the major component of the lipid bilayer of cell membranes (1). Arachidonic acid (AA), a 20-carbon fatty acid, is also present in substantial amounts in cellular membranes and is produced from LA by chain elongation and desaturation (1,2). These two PUFAs are further converted into a ) prostaglandins or thromboxanes by cyclooxygenases and/or b ) hydroxyeicosatetraenoic acids (HETEs), leukotrienes, or 13-hydroxyoctadecadieneoic acid (13-HODE) by lipoxygenases ( Fig. 1 ) (3).
The hydroxylated metabolites of LA and AA are important mediators of many physiological and pathophysiological processes (3). For instance, prostaglandins, the products of cyclooxygenases, regulate vascular homeostasis, kidney function, ovulation, and parturition (4). Although the physiological functions of HETE, the hydroxylated metabolites of AA, are not fully characterized, accumulating evidence indicates that they are involved in a wide spectrum of biological activities, such as platelet activation and aggregation, immune system activity, cell differentiation, and apoptosis (5)(6)(7). The products of cyclooxygenases and lipoxygenases have also been implicated in several forms of epithelial cell cancers, especially in prostate cancer, for which recent results indicate that they are important angiogenic and mitogenic factors (3,(8)(9)(10)(11).
Although the catabolism of these PUFAs involves several P450 enzymes, the significant contribution of glucuronidation in their clearance has been recently established (12)(13)(14)(15)(16). Indeed, several authors have reported the presence in the urine of glucuronidated PUFAs (17)(18)(19)(20). This conjugation reaction is catalyzed by UDP-glucuronosyltransferases (UGTs), a family of endoplasmic reticulum membrane-bound enzymes that transfer the glucuronic moiety from the UDP-glucuronic acid (UDPGA) to a wide variety of small lipophilic molecules carrying a functional group containing oxygen, nitrogen, or sulfur (21). Conjugation by UGT enzymes is also an important pathway of elimination for several endogenous compounds, namely steroids, thyroid hormones, retinoic acids, and bilirubin.
Based on amino acid sequence homology, UGT enzymes have been classified into two families, UGT1 and UGT2 (22). In humans, the UGT1 family is encoded by a complex of genes localized on chromosome 2q37 (23). These genes share exons 2 to 5, which are coupled to specific exons 1, leading to the expression of different transcripts that encode nine functional proteins (24). The UGT1 members are known to catalyze the conjugation of bilirubins, phenols, and estrogen hormones (25,26). The UGT2 family is divided into two subfamilies, UGT2A, which is expressed in olphactive epithelium, and UGT2B (27). The genes encoding the UGT2B members have been localized on chromosome 4q13, and each of them is composed of six specific exons (28). In humans, seven members of the UGT2B family have been characterized: UGT2B4 (29,30), UGT2B7 (31,32), UGT2B10 (33), UGT2B11 (31), UGT2B15 (28), UGT2B17 (34), and more recently UGT2B28 (35). All UGT2B cDNAs have been stably transfected in the HK293 cell line, and it was demonstrated that these enzymes and their allelic variants have conjugation activities toward carboxylic acids, bile acids, and steroid hormones, namely androgens and estrogens (35,36). However, UGT2B10 and UGT2B11 did not demonstrate conjugating activity for over 100 substrates tested (33).
The objective of this study was to demonstrate the role of UGT enzymes in the conjugation of the AA and LA metabolites. All available human UGT1A and UGT2B enzymes stably transfected in the HK293 cell line, devoid of endogenous steroid transferase activity, have been tested. Our data demonstrate that several UGT enzymes conjugate AA metabolites 5-HETE, 12-HETE, 15-HETE, and leukotriene B 4 (LTB 4 ), and the LA metabolite 13-HODE.

Glucuronidation assay using microsomal fractions
Enzymatic assays were conducted using 1 mM UDPGA, 30 M to 200 M of the different aglycons, and 5 M to 40 g of protein from microsome preparations in 50 mM Tris-HCl (pH 7.6), 10 mM MgCl 2 , 100 g/ml phosphatidylcholine, and 8.5 mM saccharolactone in a final volume of 100 l. Assays were terminated by adding 100 l of methanol and by centrifugation at 14,000 g for 1 min, as previously described (34). The screening for the reactivity of the various UGT proteins with fatty acid substrates was performed using 1 mM UDPGA for 16 h at 37 Њ C. Compounds that demonstrated reactivity with UGT proteins were subsequently reassayed in the presence of 1 mM UDPGA for 1 h with all UGT enzymes, except for UGT2B17, which was incubated for 30 min at 37 Њ C in order to obtain linear reaction rates. The microsomal fraction from HK293 cells, which do not express endogenous UGT enzymes, was used as a negative control for the glucuronidation reaction.
by guest, on July 20, 2018 www.jlr.org Downloaded from Alliance 2690 system (Waters, Milford, MA). Chromatographic separation was achieved with a Phenomenex Colombus C18 column, 5 m particles, 50 ϫ 3.2 mm (Phenomenex, Torrance, CA) using a two-solvent gradient system: A ) 1 mM ammonium formate in water; and B ) 1 mM ammonium formate in methanol. A linear gradient from 15% to 95% B was run over 6 min, held 2 min and then reequilibrated to 15% of the second gradient system over 2 min at the constant flow rate of 0.7 ml/min. The HPLC column was coupled to a Finnigan LCQ ion trap mass spectrometer equipped with an electrospray ionization source in the negative ion mode. The mass spectrometer was operated in full scan mass spectrometry (MS) mode, except in fragmentation studies where tandem MS scan was used.

K m determination
Determination of the K m values of UGT1A3 and UGT2B7 for

Effect of ADT on LTB 4 glucuronidation
Microsomal proteins from HK293 cells stably expressing UGT2B7 were incubated in the presence of 100 M LTB 4 and concentrations of ADT ranging from 0.1 M to 100 M, which represent 0.01-to 10-fold the K m value of UGT2B7 for this substrate (37).
Assays were incubated for 1 h at 37 Њ C and glucuronidation products were subsequently analyzed by liquid chromatography (LC)-MS.

Glucuronidation of arachidonic and LA metabolites by human liver microsomes
To ascertain the experimental conditions used for incubation of human liver microsomal preparations with LA and AA metabolites and the system of quantification by liquid chromatography-mass spectrometry (LC-MS), incubations were first conducted with LTB 4 and its 20-COOH metabolite, for which the formation of conjugated products by human liver microsomes has already been demonstrated (12). As illustrated in Fig. 2A , LTB 4 was detected in the supernatant at the retention time of 6.41 min when the detector was set at m/z 335-337, whereas two major metabolites corresponding to the monoglucuronide derivatives of LTB 4 ( m/z 511-513) eluted at retention times of 5.14 and 5.86 min (Fig. 2B). Our data indicate that the LTB 4monoglucuronide molecules were fragmented into the parental LTB 4 molecule and a glucuronic acid ( m/z 176) (data not shown), and that the formation of di-or triglu-  curonide of LTB 4 was not be observed. The substrate 20-COOH-LTB 4 was also conjugated by hepatic UGT enzymes and, in this case, only one metabolite was observed at retention time of 3.23 min at m/z 540-543, also corresponding to a monoglucuronide derivative (Fig. 2C, D).
Glucuronidation reaction by human liver microsomes of 5-, 12-, and 15-HETE, and 13-HODE was also assessed and, as shown in Fig. 3 . Glucuronide derivatives were detected by MS for each of the substrates tested. Except for 5-HETE and 13-HODE, which were conjugated into two distinct monoglucuronides, the two other fatty acids yielded only one product at the m/z corresponding to a monoglucuronide, and the formation of diglucuronide was not detected. Incubation of liver microsomes with 12-HETE, 15-HETE, and 13-HODE for 16 h resulted in the transformation of more than 95% of the substrates, whereas 5-HETE and LTB4 were converted by 53% and 17%, respectively.

Glucuronidation of AA and LA metabolites by stably transfected UGT enzymes in HK293
To determine which UGT enzymes are responsible for the glucuronidation of the substrates tested, incubations were performed with all recombinant UGT1 and UGT2 family members presently available ( Table 1 ). Glucuronidation of LTB 4 was observed with UGT1A1, UGT1A3, UGT1A8, and UGT2B7. Screening for glucuronidation activity toward 20-COOH-LTB 4 demonstrated that UGT1A9 is the only isoform that catalyzes conjugation of this compound. Among the UGT1A family members, UGT1A9 demonstrated wide substrate specificity, conjugating the three HETEs and 13-HODE. UGT1A1, UGT1A3, and UGT1A4 also showed broad specificity toward PUFA, conjugating 12-HETE, 15-HETE, and 13-HODE. LA and AA metabolites also appear to be good substrates for the UGT2B subfamily members, especially for UGT2B4 and UGT2B7, which conjugate all HETEs and 13-HODE. Surprisingly, UGT2B15 and UGT2B17, which are known to conjugate androgens, glucuronidate 12-HETE, and 13-HODE, respectively. Most interestingly, UGT2B10 and UGT2B11, which are considered orphan enzymes since no conjugation activity has so far been demonstrated with these enzymes, both conjugated 12-HETE, 15-HETE, and 13-HODE, whereas UGT2B10 also glucuronidated 5-HETE. Figure 4 illustrates the LC-MS analysis of the supernatant from incubation of UGT2B10 with 12-HETE, where a product at m/z corresponding to a monoglucuronide derivative of 12-HETE was detected. To ascertain that the absence of conjugation activity toward fatty acid derivatives observed with other UGT isoforms cannot be attributed to protein degradation, each UGT enzyme was incubated in the presence of eugenol. Glucuronidation toward this positive marker of glucuronidation was observed with all enzymes, with the exception of UGT2B10, UGT2B11, and UGT1A5, the latter having no identified substrate thus far (data not shown).

Kinetic analyses of AA and LA glucuronidation
To determine the extent of glucuronidation toward fatty acid metabolites, all isofoms that significantly reacted with PUFA were reassayed in linear conditions. Kinetic analyses performed with microsomal preparations of human liver and recombinant UGT enzymes demonstrated high levels of LTB 4 conjugation. The glucuronidation rates were 1,688, 117, 16, and 493 pmol·min Ϫ1 ·mg protein Ϫ1 for liver, UGT1A3, UGT1A8, and UGT2B7 microsomes, respectively (Fig. 5). Although screening analyses demonstrated that UGT1A1 conjugates LTB 4 (Table 1), the low level of conjugation rate did not allow quantification under the conditions used for kinetic analysis. Determination of UGT affinities for LTB 4 was performed using microsomal preparations of stably transfected UGT1A3 and UGT2B7. As shown in Fig. 6, K m values were 35 M and 28 M for UGT1A3 and UGT2B7, respectively, and the glucuronidation efficiency corresponding to the V max /K m ratio was 3.4 and 17.6 l·min Ϫ1 ·mg protein Ϫ1 for UGT1A3 and UGT2B7, respectively.
Because previous experiments suggested that UGT2B7 shows the broader substrate specificity and the higher velocity toward the majority of PUFAs, we retained this enzyme to investigate the glucuronidation capacities of UGT enzymes toward 5-HETE, 12-HETE, and 15-HETE. As observed with LTB 4 , UGT2B7 is an excellent conjugator of these eicosanoids, with glucuronidation rates of 88, 855, and 591 pmol·min Ϫ1 ·mg protein Ϫ1 , respectively (Fig. 7). K m values of the UGT2B7 enzyme for 5-, 12-, and 15-HETE are, respectively, 80 M, 71 M, and 52 M, leading to glucuronidation efficiencies of 1 l, 12 l, and 11 l·min Ϫ1 ·mg protein Ϫ1 .
HETE, hydroxyeicosatetraenoic acid; HODE, hydroxyoctadecadienoic acid; LTB 4 , leukotriene B 4 ; UGT, UDP-glucuronosyltransferase. To determine which UGT isoenzyme was involved, microsomal proteins (40 g) were incubated with 1 mM UDP-glucuronic acid, 200 M of substrate for 16 h at 37ЊC. Enzymatic reactions were stopped by adding methanol, and the samples were centrifuged to remove proteins. The supernatants (25 l) were analyzed by liquid chromatographymass spectrometry. (ϩ) indicates that glucuronidation activity was observed, whereas (-) indicates that no conjugation was observed.

Effect of steroids on PUFA glucuronidation
To investigate the putative interaction between steroids and PUFAs for glucuronidation by UGT2B7 isoform, increasing concentrations of the major androgen metabolite, ADT, were incubated in the presence of 100 M LTB 4 . Interestingly, glucuronidation of LTB 4 at concentrations of ADT lower than 10 M was increased, whereas a decrease of LTB 4 conversion was observed at concentrations above 15 M (Fig. 8). Up to 50% inhibition was observed at the concentration of 100 M ADT, which corresponds to 10-fold the K m value of UGT2B7 for ADT (37).

DISCUSSION
The lipoxygenase enzymes constitute a family of enzymes that metabolize PUFAs such as LA and AA into a variety of biologically active products (3). These enzymes are expressed in platelets, leukocytes, and several tissues, such as the liver, lung, mammalian tissues, prostate, and colon, and recent data indicate that their products are implicated in several forms of epithelial cell cancers (3,(8)(9)(10)(11)(39)(40)(41). It is generally believed that the inactivation of PUFA metabolites occurs in several tissues, including the liver, through oxidation processes and, as a phase II metabolism, by glucuronidation, a conversion that is also known to be present in the liver, to further facilitate their elimination from the body (17)(18)(19)(20). Conjugation by UGT expressed in the liver as a catabolic pathway is considered extremely important for the elimination of exogenous compounds (27). Recent evidence also suggests that, for endogenous substances such as steroids, thyroid hormones, and retinoic acids, specific UGT enzymes are expressed, in addition to the liver, in several tissues including the skin, lung, mammary gland, uterus, adipocytes, and prostate (26). In the present study, glucuronidation of LTB4, 20-COOH-LTB 4 , 5-HETE, 12-HETE, 15-HETE, and 13-HODE was investigated. The data showed that several UGT enzymes are capable of converting LA and AA metabolites into glucu-  ronide derivatives. Moreover, kinetic parameters of UGT enzymes toward PUFA metabolites suggest that the glucuronidation activity may be important in the pathway of PUFA catabolism. Interestingly, several of the UGT en-zymes capable of conjugating PUFA are expressed in various tissues other than the liver (26,36).
Glucuronidation of LA and AA metabolites was first ascertained using human liver microsomes, a tissue expressing most UGT enzymes, except UGT1A7, UGT1A8, and UGT1A10. All substrates tested were efficiently conjugated by liver microsomes to polar derivatives containing the glucuronyl moiety, as assessed by MS. Screening analyses demonstrated strong activities of liver UGT1A and UGT2B isoforms, since the 16 h incubation period leads   to the conjugation of more than 95% of 12-and 15-HETE, as well as of 13-HODE. Moreover, the screening analyses with stably expressed UGT enzymes in HK293 underlined the very wide substrate specificity of UGT enzymes for PUFA metabolites with substantially different chemical structures. Indeed, UGT enzymes reacted with both 18and 20-carbon substrates carrying two and four double bonds, respectively; the apparent lack of specificity is also observed for the position of the hydroxyl groups, since 5-HETE, 12-HETE, 15-HETE, and 13-HODE were conjugated by nearly the same set of enzymes. Although there is a clear redundancy in conjugation of fatty acid metabolites by UGT enzymes, screening analyses underlined subtle substrate specificity for some conjugating enzymes. For instance, UGT2B15, which shows 95% homology with UGT2B17 (34), conjugated 12-HETE exclusively, whereas the latter enzyme only conjugated 13-HODE. As observed for steroid hormones, these results suggest that the same substrate can be conjugated by several UGT enzymes, whereas each enzyme has a specific panel of substrates (26,36). The most surprising results were observed, however, with UGT2B10 and UGT2B11, which conjugate 13-HODE and almost all HETE substrates. These enzymes were screened over the past years on more than 100 substrates, including eugenol that is conjugated by all active UGT enzymes, but no substrate has so far been identified (33). Thus, we demonstrate herein for the first time that two UGT enzymes, UGT2B10 and UGT2B11, can catalyze the glucuronidation of LA and AA metabolites; in addition, several UGT enzymes that were previously considered highly specific for steroids may also be implicated in the glucuronidation of these products.
While screening analyses revealed the significant activity of UGT enzymes toward PUFA metabolites, the kinetic parameters of their conjugation support a role of UGT enzymes in their metabolism. Indeed, comparison of LTB 4 glucuronidation by UGT2B7 with that of other substrates showed high glucuronidation rates. For instance, the glucuronidation rate for LTB 4 exceeded by almost 4-fold the rate observed for ADT, the endogenous substrate of UGT2B7 with the highest glucuronidation rate observed to date (36). Interestingly, UGT2B7 glucuronidation rates for LTB 4 are similar to those of eugenol, which is generally used as a positive control for glucuronidation (36). Since kinetic analyses demonstrated that UGT2B7 has a 3-fold lower affinity for LTB 4 than for ADT (36,37), the resulting LTB 4 glucuronidation efficiency, as expressed by the V max -K m ratio, was 17.6 l·min Ϫ1 ·mg protein Ϫ1 , which was in the range of the glucuronidation efficiency observed for steroids. Results obtained with UGT1A3 also demonstrated very high glucuronidation rates for LTB 4 and K m values within the micromolar range. To gain more information on the glucuronidation rates of PUFA metabolites, UGT enzymes that showed the highest activity for 5-, 12-, and 15-HETE during screening analyses were investigated to determine the kinetic parameters of glucuronidation. As observed with LTB 4 , glucuronidation rates were higher than that for steroid hormones, especially for 12and 15-HETE, which have V max values of 855 and 591 pmol·min Ϫ1 ·mg protein Ϫ1 , respectively; however, affinity of the UGT2B7 enzyme for the tested substrates was relatively low; therefore, glucuronidation efficiencies were similar to those observed with steroid hormones. The levels of glucuronidation efficiencies observed in the present study are also in agreement with data reported previously by Jude et al. of LA conjugation by UGT2B7 (14).
In the human prostate, 5-HETE and 13-HODE promote cell proliferation, whereas 12-HETE and 15-HETE may regulate angiogenesis and prostate cell differentiation, respectively (3,10,11). Our previous studies revealed that UGT2B4, UGT2B10, UGT2B11, UGT2B15, and UGT2B17 are expressed in the prostate (36,42); therefore the present data suggest that prostate tissue has the capacity to conjugate the lipoxygenase products. In addition, some of these UGT enzymes, namely UGT2B15 and UGT2B17, can also inactivate dihydrotestosterone and its metabolites formed in the prostate (26). As for some PUFA metabolites, androgens also play a determining role in prostate differentiation and development, and stimulate the growth of prostate cancer cells. Therefore, UGT enzymes expressed in the prostate could be implicated in the metabolism of two distinct classes of endogenous compounds. Our data further reinforce the concept that several tissues possess the enzymatic machinery to produce and inactivate biologically active products (26,43,44). In addition, since it was previously demonstrated that the expression of UGT enzymes in LNCaP cells could be modulated by several factors, such as androgens, growth factors, and cytokines, it is reasonable to believe that concentrations of PUFAs metabolites in tissues such as human prostate may also be altered by changes in inactivating enzyme activity (45,46). The dual action of UGT enzymes on two classes of biologically-active products is further supported by competition analyses that demonstrated that both steroids and PUFAs compete in vitro for the same binding sites or two overlapping sites. In this experiment, it was also interesting to observe an increase of LTB 4 conjugation by UGT2B7 at low concentrations of ADT. A homotropic activation of glucuronidation activity at low concentrations of substrates was recently reported for estradiol conjugation at position 3 by UGT1A1 (47), whereas this effect was not observed for the glucuronidation of estradiol at position 17 by UGT2B7. Although the phenomenon may be an artifact of the in vitro system used, further studies will be needed to evaluate this conjugation in vivo.
The expression of UGT enzymes capable of conjugating leukotrienes in tissues such as the liver and lung also suggests a role of these enzymes in the catabolism of both systemic and locally produced leukotrienes, and a possible implication of UGT enzymes in pathologies such as asthma and allergic airway response through regulation of their levels. Although leukocytes, especially neutrophils, are known to efficiently catalyze the degradation and inactivation of LTB 4 , the role of the liver would also be of major importance, since this tissue is also responsible for the elimination of LA and AA metabolites released into systemic circulation (48)(49)(50).
In summary, our data demonstrate that several UGT en-by guest, on July 20, 2018 www.jlr.org Downloaded from zymes are capable of conjugating LA and AA metabolites. Since this conjugating activity is expressed in several tissues other than the liver, it is reasonable to believe that UGT enzymes may serve to maintain homeostasis of physiologically important endogenous substances and/or to protect from deleterious high concentrations.