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Research ArticleArticle

Dog UDP-Glucuronosyltransferase Enzymes of Subfamily 1A: Cloning, Expression, and Activity

Johanna Troberg, Erkka Järvinen, Maria Muniz, Nina Sneitz, Johanna Mosorin, Marja Hagström and Moshe Finel
Drug Metabolism and Disposition January 2015, 43 (1) 107-118; DOI: https://doi.org/10.1124/dmd.114.059303

Abstract

Understanding drug glucuronidation in the dog, a preclinical animal, is important but currently poorly characterized at the level of individual enzymes. We have constructed cDNAs for the 10 dog UDP-glucuronosyltransferases of subfamily 1A (dUGT1As), expressed them in insect cells, and assayed their activity as well as the activity of the nine human UGT1As, toward 14 compounds. The goal was to find out whether individual dUGT1As and individual human UGT1As have similar substrate specificities. The results revealed similarities but also many differences. For example, similarly to the human UGT1A10, dUGT1A11 exhibited high glucuronidation activity toward the 3-OH of 17-β-estradiol, 17-α-estradiol, and ethinylestradiol, and also conjugated the drug entacapone. Unlike the human UGT1A10, however, it failed to catalyze considerable rates of R-propranolol, diclofenac, and indomethacin glucuronidation. The estrogen glucuronidation assays revealed that dUGT1A8 and dUGT1A10 have a capacity to catalyze the formation of (linked) diglucuronides, an activity no human UGT1A exhibited. dUGT1A2-dUGT1A4 are homologs of the human UGT1A4, but none of them catalyzed N-glucuronidation of dexmedetomidine. Contrary to the human UGT1A4, however, dUGT1A2-dUGT1A4 catalyzed indomethacin and diclofenac glucuronidation. It may be concluded that, perhaps with the exception of UGT1A6, high similarities in substrate specificity between individual dog and human UGTs of subfamily 1A are rare or partial. Activity assays with liver and intestine microsomes of both dog and human further revealed interspecies differences, particularly in glucuronidation rates. In the dog, the microsomes assays also strongly suggested important roles for dUGTs of other subfamilies, mainly in the liver.

Introduction

Safety evaluation of a new drug candidate in preclinical animals before first human exposure is a necessary stage in the drug development process. These tests include at least two preclinical species, a rodent and a nonrodent; the latter is often a dog (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM078933.pdf). Although these tests are important, differences in drug metabolism between preclinical animal species and humans might affect considerably safety evaluation in different ways. Better knowledge of the enzymes that catalyze the tested reactions in different preclinical animals may allow improved interpretation of the results and, at the same time, contribute to the development of veterinary drugs for different domestic animals.

Glucuronidation of endogenous compounds and xenobiotics including drugs either directly or of their phase I metabolites is catalyzed by UDP-glucuronosyltransferase enzymes (UGTs) and plays an important role in their biotransformation and pharmacokinetics (for reviews, see King et al., 2000; Tukey and Strassburg, 2000). The UGTs catalyze the transfer of the glucuronic acid moiety from the cosubstrate UDP-glucuronic acid (UDPGA) to a nucleophilic group on the acceptor molecule, mostly a hydroxyl or different amino groups. There are 19 different human UGTs that are divided into subfamilies 1A, 2A, and 2B (Mackenzie et al., 2005). In addition, there are a few other closely related sugar transferases that mainly catalyze the transfer of other sugar moieties (Rowland et al., 2013), but they will not be further discussed in this work.

About half the human UGTs belong to subfamily 1A, which is encoded by a single large gene, ugt1a. Due to exon sharing, the mature mRNA for each UGT1A contains a different (but homologous) exon 1 and the same four exons 2–5 that are shared by all the UGTs of this subfamily (splice variants among exons 2–5 are beyond the scope of this study). The first exon encodes the N-terminal half of the protein, whereas the shared exons together encode the C-terminal half of the UGT (Mackenzie et al., 2005).

Glucuronidation activity in dog liver and intestine microsomes (DLM and DIM, respectively) was previously examined and revealed similarities, alongside many differences, in substrate specificity and glucuronidation rates in comparison with human liver and intestine microsomes microsomes (HLM and HIM, respectively) (Soars et al., 2001a; Kaivosaari et al., 2011; Furukawa et al., 2014). The dog UGT1A6 was earlier cloned, sequenced, expressed, and examined for the glucuronidation of many compounds, revealing high similarity to the human UGT1A6 (Soars et al., 2001b). Another dog UGT, named dUGT2B31, was later cloned, expressed, and characterized by the same research group, but it was difficult to identify a clear and single human UGT2B enzyme as its homolog (Soars et al., 2003).

The complete dog genome was earlier sequenced (Kirkness et al., 2003; Lindblad-Toh et al., 2005) and that data, in combination with the knowledge of exon sharing in the ugt1a gene, guided the bioinformatic analysis of the dog genome, yielding cDNA sequences for the different dUGT1A enzymes (Li and Wu, 2007). There is, however, a minor mixup in the names of individual human and dog UGT1As because of the differences in pseudogene content and exon 1s naming order (Fig. 1). In addition, the sequence identities between individual exon 1s of the dog and human UGT1As are not high enough to “pair” individual UGT1As among the groups dUGT1A2–dUGT1A4 and the human UGT1A3–UGT1A5 as well as dUGT1A7–dUGT1A11 and the human UGT1A7–UGT1A10 (Fig. 2). Therefore, we have followed the names given by Li and Wu (2007) for the dog UGT1As, but the common names for the human UGT1As.

Fig. 1.
Fig. 1.

Schematic view of the arrangement of the exons in the dog and human ugt1a genes. The constant exons 2–5 are included in each dUGT1A/UGT1A, whereas different UGT1As contain different exon 1s. The first human exon 1s A2, A13, A11, and A12 are pseudogenes. Note the differences in the order/names of the variable exons between the two different ugt1a genes.

Fig. 2.
Fig. 2.

A phylogenetic tree, based on the amino acid sequence identity degree of the N-terminal domain, the UGTs segments that are encoded by the respective exon 1s, between the human and dog UGT1As. The tree was drawn using the free online Phylogeny.fr service (http://www.phylogeny.fr; Dereeper et al., 2008) The blue-colored branches highlight the dog UGT1As, and the red-colored branches show human UGT1As, indicated here as hUGTs.

The main objective of this study was to explore interspecies specificity in UGT1As at the level of individual enzymes. To this end, we have cloned, expressed, and assayed the activity of the 10 dUGT1As, using 14 substrates that are known to be human UGTs substrates even though some of them such as testosterone and epitestosterone are mainly substrates of human UGTs of subfamilies 2A and 2B (Sten et al., 2009). The dUGT1As activities were compared with the activities of the nine human UGT1As, which were expressed and assayed in the same way. In addition, the glucuronidation of the same compounds in liver and intestine microsomes from both dog and human was determined to obtain a broader picture of the glucuronidation capacity in these tissues and the probable contribution of UGTs of other subfamilies to this activity.

Materials and Methods

Materials.

UDP-α-D-glucuronic acid (UDPGA, ammonium salt), 4-methylumbelliferone (4-MU), 4-MU-β-d-glucuronide, 17-α-estradiol (epi-E2), 17-β-estradiol (E2), E2-β-d-3-glucuronide (E2-3-glucuronide), E2-β-d-17-glucuronide (E2-17-glucuronide), 17-α-ethinylestradiol (ethinylestradiol), epitestosterone, bilirubin, diclofenac, indomethacin, R-propranolol, S-propranolol, sodium phosphate monobasic dihydrate, and disodium hydrogen phosphate were purchased from Sigma-Aldrich (St. Louis, MO). Entacapone, dexmedetomidine, and levomedetomidine were kindly provided by Orion Pharma Corporation (Espoo, Finland). Entacapone-β-d-glucuronide was synthesized in our laboratory (Luukkanen et al., 1999). Testosterone was from TCI Europe (Zwijndrecht, Belgium), and magnesium chloride hexahydrate and perchloric acid (PCA) were from Merck (Darmstadt, Germany). Formic acid was from Riedel-deHaën (Seelze, Germany). Dimethylsulfoxide (DMSO), acetonitrile, methanol, and acetic acid were high-pressure liquid chromatography (HPLC) grade. Alamethicin was obtained from A.G. Scientific (San Diego, CA), and the oligonucleotide primers (Table 1) were from Oligomer (Helsinki, Finland).

View this table:
TABLE 1

Oligonucleotides for the dUGT1As cloning

Inserted restriction site sequences are underlined.

Pooled HLM, HIM, and pooled DLM (male beagle) were bought from BD Biosciences (Franklin Lakes, NJ), whereas dog pooled intestinal microsomes (DIM, male beagle) were from Xenotech, LLC (Lenexa, KS). Dog liver cDNA (C1734149) was purchased from Biochain (Newark, CA). The genomic DNA of beagle dogs was a generous gift from Dr. Hannes Lohi, University of Helsinki.

Cloning and Expression of Recombinant dUGT1As.

The cloning strategy for constructing complete cDNAs for the 10 different dog UGT1A enzymes was based on amplifying one “shared segment” made of correctly spliced exons 2–5 from hepatic cDNA using oligonucleotides no. 1 and 2 (Table 1) and ligating to it the different exons 1s of each dUGT1A. To facilitate an in frame ligation of the 3′-end of the amplified exon 1 to the 5′-end of the “shared segment,” an EcoR1 restriction site was generated without changing an amino acid (silent mutagenesis) at the 5′-end of exon 2 (no internal EcoR1 site was found in exons 2–5).

Each of the 10 full-length first (variable) exons was amplified from genomic beagle DNA, by itself or as a group (see below), using 5′ polymerase chain reaction (PCR) primers (indicated by F in Table 1) that were designed based on the upstream flanking regions sequences in gene bank (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_PROG_DEF=megaBlast&BLAST_SPEC=OGP__9615__10726) and 3′ primers (indicated by R, Table 1) that were designed using the sequences in Li and Wu (2007). Most of the R primers also contained an EcoR1 restriction site for in frame ligation to the EcoR1 site at the beginning of the shared segment (see Table 1 for PCR primer sequences). An upstream restriction site was included in the F primers for the first exons to make subsequent subcloning simpler (the selected restriction site in each case is indicated in the primer’s name; see Table 1).

Two groups of exon 1s of the dUGTs, d1A2–d1A4, and d1A8–d1A11, have very high degree of sequence identity within their exon 1s segments (see also Fig. 2). Due to this, in these cases, instead of designing specific oligonucleotides for each of these UGT genes, we used F primers that are suitable for some or all the members in these very-high-homology groups (Table 1). The resulting mixed amplified PCR products were subcloned, and single colonies were picked and cultured for plasmid purification without knowing which dUGT1a they encode. Only then, after DNA sequencing, did it become clear which clone contained which cDNA. There was another complication in the case of dUGTs 1A2–1A4: the presence of an internal EcoR1 site at about the middle of exon 1. To overcome this internal site, we inserted an Nde1 restriction site downstream the internal EcoR1 site (using oligonucleotides no. 8 and 9, Table 1) and subcloned the two parts of these exons, the BamH1-Nde1 fragment and the Nde1-EcoR1 fragment together, as a 3-fragment ligation in which the third was the vector made from the exons 2–5 segment in pFastBac-XHC (the vector with the fusion peptide before the insertion of the C-terminal of dUGT1A is described in Kurkela et al., 2003) that was digested with BamH1 and EcoR1.

After cDNA construction within the modified pFast-Bac vector, pFB-XHC (Kurkela et al., 2003), the shuttle vectors (bacmids) and recombinant baculoviruses were prepared using the Bac-to-Bac system (Invitrogen, Carlsbad, CA). The different recombinant His-tagged dog UGTs were produced in sf9 insect cells, and cell homogenates were used as the study material in the activity assays. Total protein concentrations and relative expression levels of each recombinant dUGT were determined as described previously elsewhere (Zhang et al., 2012a).

Because the first batch of cell homogenates for each dUGT was not sufficient for all the assays, we prepared a second one and determined the relative expression level of each dUGT in that batch, too. The second batches of dUGTs cell homogenates were mainly used for the assays of indomethacin, diclofenac, R- and S-propranolol glucuronidation. The relative expression levels for the two batches of cell homogenates expressing recombinant dog UGTs were as follows (the expression levels in the second batches are given square brackets): d1A1, 5.4 [2.9]; d1A2, 7.9 [4.9]; d1A3, 12.6 [3.4]; d1A4, 12.3 [7.4]; d1A6, 9.3 [6.4]; d1A7, 14.2 [6.1]; d1A8, 3.7 [1.0]; d1A9, 9.1 [14.1]; d1A10, 1.6 [1.6]; and d1A11, 3.4 [8.1]. The human recombinant UGTs were expressed and prepared as enriched insect cell membranes as previously described elsewhere (Zhang et al., 2012a), and their relative expression levels were 1A1, 2.1; 1A3, 19.5; 1A4, 5.2; 1A5, 17.0; 1A6, 2.5; 1A7, 21.5; 1A8, 11.0; 1A9, 5.2; and 1A10, 1.3. These relative expression values for both the dog and human UGTs were determined using the same reference values to allow direct comparisons between them, and they were used to “normalize” the activity of different UGTs or to correct the measured glucuronidation rates, per milligram of protein, according to the relative expression level of each enzyme.

Glucuronidation Assays.

The substrates (see Table 2) in methanol were added to the incubation tubes, followed by evaporation of the methanol and dissolution of the residue in the reaction mixture, containing 50 mM phosphate buffer, pH 7.4, 10 mM MgCl2, and the enzyme source. The assay conditions were adjusted to avoid consumption of more than 30% of the substrate during the incubation. To this end, in assays using recombinant dog UGTs cell homogenates, the total protein concentration was 1.0–2.2 mg/ml; in assays using recombinant human UGTs in membrane preparations, the total protein concentration was lower, 0.1–0.2 mg/ml. When pooled liver or intestine microsomes of either dog or human were the enzyme source, the protein concentration was 0.1–0.2 mg/ml, and the reaction mixtures also contained 50 µg/mg alamethicin per total protein (Walsky et al., 2012). In the cases of indomethacin and diclofenac glucuronidation reactions, the pH of the phosphate buffer was adjusted to 6.0 to reduce acyl migration, as previously reported elsewhere (Zhang et al., 2012b). The bilirubin glucuronidation assays were performed avoiding light exposure to minimize substrate isomerization.

View this table:
TABLE 2

Glucuronidation assay conditions

The ready mixtures were first preincubated at 0°C for either 15 minutes (recombinant dog and human UGTs) or 30 minutes (dog and human pooled microsomes), followed by 5 minutes at 37°C, before starting the reaction by the addition of UDPGA to a final concentration of 5 mM, in 100 µl reaction volume. Enzyme reactions were performed at 37°C for 5–120 minutes and terminated as described in Table 2 and in the supplemental materials (including Supplemental Fig. 1). After the reaction termination, the samples were transferred to −20°C for an hour and then centrifuged at 16,000g for 10 minutes. Aliquots of the supernatants were analyzed by HPLC. The assays were performed in triplicate and included control samples without UDPGA.

As presented below, in assays of estrogen glucuronidation by dUGT1A8 and dUGT1A10, a new glucuronide peak was detected that was not seen in assays with the other dUGT1As or the human UGT1As. For further examination of this glucuronide, which turned out to be estrogen diglucuronides (or linked glucuronides; Argikar, 2012), dUGT1A8 and dUGT1A10 were also incubated with either of the two E2-monoglucuronides, namely, E2-3-glucuronide or E2-17-glucuronide, as the sole aglycone substrate. These incubation reactions contained either 75 µM of E2-3-glucuronide or 125 µM of E2-17-glucuronide without DMSO. Otherwise, the reaction conditions and HPLC analyses were performed as described for estradiol (Tables 2 and 3).

View this table:
TABLE 3

Analytic conditions and methods for HPLC glucuronide separation

The eluents were acetonitrile (B) and 50 mM phosphate buffer pH 3.0 (A) in all methods, except that in the R- and S-propranolol and bilirubin chromatographic methods 0.1% formic acid in water was used as eluent A. The column temperature and the flow rate were 40°C and 1 ml/min, respectively.

Analytic Methods.

HPLC analyses were performed using Agilent 1100 series equipment with multiple wavelengths UV detector and fluorescence detector, connected to a fraction collector (Agilent Technologies, Palo Alto, CA). A Poroshell 120 EC-C18 column (4.6 × 100 mm, 2.7 µm; Agilent Technologies) was used for glucuronide(s) and substrate separations. Details of the gradient HPLC runs are presented in Table 3. The resulting chromatograms were examined with Agilent ChemStation software and further analyzed with GraphPad Prism, version 5.04 for Windows (GraphPad Software, San Diego, CA).

Quantification of the glucuronides in the assays was based on standard curves generated with authentic standards, when such standards were available (i.e., entacapone-glucuronide, 4-MU-glucuronide, E2-3-glucuronide, and E2-17-glucuronide). In other cases, a close estimate of the generated glucuronide was employed by using the UV absorbance of the aglycone to prepare the standard curve (Court, 2005). If an authentic standard was not available and when fluorescence was used to detect low glucuronidation rates—namely, in the cases of epi-E2, ethinylestradiol, and R- and S-propranolol—the standard curve was generated for the glucuronide by correlating its fluorescence to its UV signal (in reactions where much of it was produced) and to a standard curve prepared with the UV absorbance of the aglycone. The limits of determination and quantification were determined by visual evaluation from chromatograms of the samples and controls without UDPGA or without substrate.

Liquid chromatography with mass spectrometry (LC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analyses were performed in few cases to further characterize the glucuronide. Fractions from the peaks of interest were subjected to Waters Acquity UPLC equipped with Waters Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm) (Waters, Milford, MA). The chromatographic separation was performed using 0.1% formic acid in water as eluent A and 0.1% formic acid in acetonitrile as eluent B, at a constant flow of 0.4 ml/min, column temperature of 26°C, and the following gradient: 0–0.5 minutes, 5% B; 0.5–4 minutes, 5→95% B; 4–4.6 minutes, 95→5% B; and 4.6–6 minutes, 5% B. Mass spectrometric measurements were performed using Waters Xevo Q-TOF (quadrupole time-of-flight) MS with negative mode electrospray ionization. The scanning modes were MS scan and product ion scan, with mass range of 100–1000 m/z. The parameters for capillary voltages, sample cone, and extraction cone were 2100 V, 21.0 V, 2.2 V, respectively. The source temperature was 100°C, and the desolvation temperature was 200°C. Nitrogen (Aga, Helsinki, Finland) was used as desolvation gas at a flow rate of 800 l/h and 0 l/h for cone, and the collision energy was 10–30 eV.

Results

dUGT1A Cloning and Expression.

To clone and express all the dUGT1As as individual recombinant proteins, even when having their DNA sequences (from Li and Wu, 2007), one should obtain the respective mRNA, total RNA, or cDNA from a tissue in which each gene is expressed. Hepatic beagle dog cDNA is likely to contain cDNAs for some of the dUGT1As but not for the extrahepatic enzymes. Nevertheless, relying on the exon sharing system in this subfamily, having beagle dog hepatic cDNA and genomic DNA is a suitable source for constructing cDNAs for all the dUGT1As, including the extrahepatic ones, following the approach we previously used to construct full-length cDNAs for the extrahepatic human UGTs 1A7, 1A8, 1A10 without having RNA from the tissues in which they are expressed (Kuuranne et al., 2003). Because it was previously shown that full-length dUGT1A6 is expressed in the liver (Soars et al., 2001b), we used hepatic dog cDNA library to clone the “shared (or constant) segment” (Fig. 1), correctly spliced exons 2–5 that together encode the C-terminal domain of all the dUGT1As. The variable exon 1s were amplified from genomic DNA and ligated, in frame, to the shared segment, as described in the Materials and Methods section.

In addition, a fusion peptide ending with a His-tag was added to the C-terminal end of the shared segment to enable determination of relative expression level of the dUGT1As and to correct (normalize) their glucuronidation rates according to it. A similar normalization of rates, using the same references, was performed for the human UGT1As, allowing direct comparisons of the activity rates between all the tested UGTs in this study.

The names of the individual dUGT1As in this study are according to Li and Wu (2007) (except the use of “A” here for the dog as well, instead of “a,” following recommendation of Dr. P. Mackenzie); however, it should be noted that they do not fully correspond to the human UGT1A names. As shown in Fig. 1, there are differences in the number and location of pseudogene in the large ugt1a gene as well as differences in the order in which the first exons were numbered, once their numeric value is larger than 7 (Fig. 1). In addition, and perhaps more important than the names inconsistency between the exon 1s of the dUGT1As and the human UGT1As, while there is a degree of protein sequence identity between corresponding dog and human UGT1As, it is not high enough to assume identical activities. As can be seen from the phylogenetic analysis of the dog and human UGT1As (Fig. 2), the degree of sequence identity among UGTs d1A2–d1A4 is higher than between any of them to any of the human UGTs 1A3–1A5 and vice versa. A similar situation is seen between UGTs d1A7–d1A11 on the one hand to the human 1A7–1A10 on the other (Fig. 2). Taken together, these differences prompted us to examine activity similarity, mainly qualitatively, by testing all the dog and human UGT1As in each assay regardless their name, location in the ugt1a gene, or degree of sequence identity.

Glucuronidation Activity Assays.

The recombinant dUGT1As in enriched insect cell homogenates were subjected to glucuronidation activity assays toward 14 compounds, including several diastereomers and enantiomers which were assayed as a pure stereoisomer not as a mixture. The human UGT1As in enriched insect cells membranes were subjected to assays with the same 14 compounds. The glucuronidation rates of all the recombinant UGTs were normalized or corrected according to their relative expression levels per milligram of total protein in the sample (see Materials and Methods). Each substrate was assayed at a single concentration (Table 2), and the determined glucuronidation rates are presented for both the dog (upper panel in the figures) and the human (lower panel) UGT1As in each case.

Bilirubin and Estrogens.

Bilirubin glucuronidation, an important activity of the human UGT1A1 (Bosma et al., 1992; Zhang et al., 2007), is also specifically catalyzed by dUGT1A1 among the dUGT1As (Fig. 3). The results suggest that the bilirubin glucuronidation activity of dUGT1A is lower than the corresponding activity of the human UGT1A1 (Fig. 3), a result that is supported by the bilirubin glucuronidation in microsomes.

Fig. 3.
Fig. 3.

Bilirubin glucuronidation rates by dog UGT1As (upper panel) and human UGT1As (lower panel). The rates are corrected for relative expression level (see Materials and Methods), using the second batch of dUGT1A8 as a reference for both the dog and human UGT1As.

Another activity that was largely regarded as UGT1A1 specific as far as the human UGTs are concerned is ethinylestradiol glucuronidation (Zhang et al., 2007), although later we showed that UGT1A10 also catalyzes this activity (Höglund et al., 2011). Contrary to the bilirubin glucuronidation assay results, dUGT1A1 was nearly inactive in ethinylestradiol glucuronidation (Fig. 4), whereas the results for the human UGT1As were in line with previous reports. The inactivity of dUGT1A1 in ethinylestradiol glucuronidation does not mean that this drug is not glucuronidated in the dog, however. The results demonstrate that d1A10 and d1A11 catalyze ethinylestradiol glucuronidation at the 3-OH at much higher rates than either the human 1A1 or 1A10 (Fig. 4). It is also likely that one or more dUGT(s) of other subfamilies catalyze ethinylestradiol glucuronidation at the 17-OH.

Fig. 4.
Fig. 4.

Ethinylestradiol glucuronidation rates by dog UGT1As (upper panel) and human UGT1As (lower panel). See the legend to Fig. 3 for further details.

Estradiol (17-β-estradiol, E2) glucuronidation at the 3-OH is often used as a probe activity for human hepatic UGT1A1 (Court, 2005), and we have previously shown that the human intestinal UGT1A10 catalyzes this activity at much higher rates (Itäaho et al., 2008) (note that the activity of the commercially available human UGT1A10 is low; Zhu et al., 2012). Our results suggest that the E2 glucuronidation rate of dUGT1A1 is very low. The dog does not have a single UGT1A with a high rate of E2-3-glucuronide production, but several enzymes that catalyze this activity at lower rates than the human UGT1A10 (Fig. 5). On the other hand, many dUGT1As catalyze the formation of E2-3-glucuronide at considerable rate; a clear outcome of this study is that most dUGT1As catalyze E2 preferably at the 3-OH. In addition, low rates of E2-17-glucuronide formation (high rates of this reaction, at least in human, are mainly catalyzed by UGTs of subfamily 2B; Itäaho et al., 2008) were detected in some cases, which may be interesting because they reveal some similarity between the dog dUGT1A2 and dUGT1A3 to the human UGT1A3 and UGT1A4 (Fig. 5).

Fig. 5.
Fig. 5.

Estradiol (17-β-estradiol, E2) glucuronidation rates by dog UGT1As (upper panel) and human UGT1As (lower panel). The bars mostly represent the rate of estradiol-3-glucuronide formation. Bars representing the formation of estradiol-17-glucuronide are marked by “17” on their top. See the legend to Fig. 3 for further details.

Epiestradiol, 17-α-estradiol (epi-E2), is a diastereomer of E2, and the different configuration at C17 was shown to strongly affect the activity of several human UGTs, mainly of subfamily 2B, in several ways (Itäaho et al., 2008). The present results show that it also affects the activity of some dog UGT1As, particularly it decreases the activity of dUGT1A10 with respect to dUGT1A7 and dUGT1A11 (Fig. 6) in comparison with the corresponding E2 glucuronidation rate of each of these dUGT1As (Fig. 5).

Fig. 6.
Fig. 6.

Epiestradiol (17-α-estradiol, epi-E2) glucuronidation rates by dog UGT1As (upper panel) and human UGT1As (lower panel). See the legend to Fig. 5 for further details.

Estrogen Linked-Diglucuronide Formation.

While we were performing the chromatographic analyses of the different activity assays of the recombinant dUGT1As as well as dog liver and intestine microsomes with one of the three estrogen substrates, E2, epi-E2, and ethinylestradiol, we noticed that when the tested enzyme was dUGT1A8 or dUGT1A10, another small and earlier eluting peak appeared that was not detected in the control samples that were incubated in the absence of UDPGA. It was previously shown by Murai et al. (2005) that, under suitable assay conditions, dog liver microsomes could produce diglucuronides of several steroids. The detailed description presented in their study used MS for determining whether the product is a diglucuronide (linked diglucuronide, according to the terminology of Argikar, 2012) or a bis-glucuronide. Our LC-MS/MS analysis yielded the value previously assigned for two glucuronic acid moieties linked to each other, indicating that the first eluting peak was a linked diglucuronide. Hence, in a clear and significant advance with respect to the earlier studies that were performed using DLM that contained many different UGTs (Murai et al., 2005), we have now used recombinant dUGT1As and identified two individual enzymes that can catalyze such reactions, dUGT1A8 and dUGT1A10 (the results in Fig. 7 were obtained with dUGT1A8).

Fig. 7.
Fig. 7.

HPLC chromatograms (fluorescence detection) showing the results of E2 glucuronidation by dUGT1A8 and control incubation performed in the absence of UDPGA (see Materials and Methods and Table 3).

It was also previously shown that the true substrates for estrogen diglucuronide synthesis by DLM were the respective monoglucuronides, not the aglycone (Murai et al., 2005). We have examined this with the recombinant dUGT1A8 and dUGT1A10 by use of E2-glucuronide as the substrate in the absence of E2. The results were interesting and rather surprising: when E2-3-glucuronide was the substrate, a single linked diglucuronide was generated (Fig. 8A), a diglucuronide with the same retention time as the diglucuronide that was formed when the substrate was E2 (Fig. 7A). However, when E2-17-glucuronide was the substrate, the results revealed a single new glucuronide peak with a higher retention time (Fig. 8C). LC-MS and LC-MS/MS analyses of this new glucuronide peak (Fig. 8C) clearly showed that it, too, is a linked estradiol diglucuronide, a result that is in full agreement with the elution order of the different diglucuronides in the previous study (Murai et al., 2005). This strongly suggests that the peak in Fig. 8C was linked E2-17-diglucuronide.

Fig. 8.
Fig. 8.

HPLC chromatograms (fluorescence detection) showing the results of incubations of dUGT1A8 with E2-3-glucuronide in either the (A) presence or (B) absence of UDPGA or its incubation with E2-17-glucuronide, in either the (C) presence or (D) absence of UDPGA. See Materials and Methods and Table 3 for further details.

Medetomidines, Propranolols, Diclofenac, and Indomethacin.

Turning from estrogens and bilirubin to different types of molecules, we have examined the glucuronidation of dexmedetomidine and levomedetomidine by dUGT1As. These compounds are substrates for the human UGT1A4 and UGT2B10, enzymes that mostly catalyze N-glucuronidation reactions (Kaivosaari et al., 2008). Glucuronidation activity toward both medetomidines was found in DLM, in agreement with the previous report (Kaivosaari et al., 2008), but none of the dUGT1As appeared to catalyze the glucuronidation of either dexmedetomidine or levomedetomidine (Figs. 9 and 10, respectively). These results may suggest that in the dog both dexmedetomidine and levomedetomidine are catalyzed by one or more UGTs of subfamily 2, as discussed herein.

Fig. 9.
Fig. 9.

Dexmedetomidine glucuronidation rates by dog UGT1As (upper panel) and human UGT1As (lower panel). See the legend to Fig. 3 for further details. The G1 and G2 marks in the human samples indicate two different glucuronides (Kaivosaari et al., 2008).

Fig. 10.
Fig. 10.

Levomedetomidine glucuronidation rates by dog UGT1As (upper panel) and human UGT1As (lower panel). See the legend to Fig. 9 for further details.

Diclofenac and indomethacin carry a carboxylic acid that undergoes glucuronidation, and we have previously reported that carrying diclofenac and indomethacin glucuronidation reactions under conditions of lower pH (pH 6.0 rather than pH 7.4) strongly stimulates the glucuronidation activity of the human UGT1A10 toward both drugs, making it by far the most active human UGT of subfamily 1A in diclofenac and indomethacin glucuronidation (Zhang et al., 2012b). Testing diclofenac and indomethacin glucuronidation by the dUGT1As was expected both to reveal their activity toward carboxylic acid-containing compounds and to support or reject the similarity of dog dUGT1A11 to human UGT1A10, a similarity that appeared from its high activity toward the 3-OH of different estrogens (Figs. 4–6). The results with diclofenac and indomethacin suggest, however, that as far as the glucuronidation of these drugs is concerned, there is a significant difference between dog dUGT1A11 and human UGT1A10 (Figs. 11 and 12). In the dog, at least under assay conditions (pH 6.0), it is UGTs d1A2 and d1A3 that exhibit relatively higher activity toward both these drugs, whereas dUGT1A4 also catalyzes indomethacin glucuronidation at a relatively high rate (Fig. 12). It may be added here that the low but clearly detectable activity of human UGT1A3 in diclofenac and indomethacin glucuronidation (Figs. 11 and 12, respectively; Zhang et al., 2012b) also suggests minor similarities among the differences in the dog and human UGT1As that catalyze these reactions.

Fig. 11.
Fig. 11.

Diclofenac glucuronidation rates by dog UGT1As (upper panel) and human UGT1As (lower panel). See the legend to Fig. 3 for further details and Materials and Methods for the incubation conditions.

Fig. 12.
Fig. 12.

Indomethacin glucuronidation rates by dog UGT1As (upper panel) and human UGT1As (lower panel). See the legend to Fig. 3 for further details and Materials and Methods for the incubation conditions.

4-Methylumbelliferone, Entacapone, R- and S-Propranolol, Testosterone, and Epitestosterone.

In the first study on dUGT1A6, it was shown that it catalyzes 4-MU but at much lower rate than the human UGT1A6 (Soars et al., 2001b). In our study, using a different expression system and lower substrate concentration, the glucuronidation rates of 4-MU by dog dUGT1A6 and human UGT1A6 were similar (Fig. 13). Interestingly, among the dog UGT1As, dUGT1A6 is significantly more active than any other dUGT1A in 4-MU glucuronidation, whereas in the human the 4-MU glucuronidation activity of UGT1A10 is as high, if not somewhat higher, than the activity of UGT1A6 (Fig. 13).

Fig. 13.
Fig. 13.

4-methylumbelliferone (4-MU) glucuronidation rates by dog UGT1As (upper panel) and human UGT1As (lower panel). See the legend to Fig. 3 for further details.

Entacapone is a very good substrate for the human UGT1A9 (Lautala et al., 2000) but is also glucuronidated by the human UGTs 1A7, 1A8, and 1A10 (Luukkanen et al., 2005). Testing the dUGT1As for entacapone glucuronidation revealed that one enzyme, dUGT1A11, catalyzes this reaction at relatively high rate, even if still much lower than the rate of entacapone glucuronidation by the human UGT1A9 (Fig. 14).

Fig. 14.
Fig. 14.

Entacapone glucuronidation rates by dog UGT1As (upper panel) and human UGT1As (lower panel). See the legend to Fig. 3 for further details.

As a final attempt in this study, to support or reject similarity in substrate specificity between dUGT1A11 and the human UGT1A10 or any of the human UGT1As in the 1A7–1A10 group, we tested the glucuronidation of R-propranolol and S-propranolol by the dog and human UGT1As. The propranolol enantiomers were previously shown to be useful in exposing opposite stereoselectivity in the human UGT1A9 and UGT1A10 (Sten et al., 2006), so we anticipated that dUGT1A10 and dUGT1A11 will glucuronidate one of them, if not both. The results were different, however. Only dUGT1A7 exhibited meaningful glucuronidation rate and only toward R-propranolol, with dUGTs 1A1–1A3 exhibiting low but measurable rates of this activity (Fig. 15). In the case of S-propranolol, the preferred propranolol enantiomer of the human UGT1A9, only barely detectable activity was exhibited by the dUGT1As (Fig. 16).

Fig. 15.
Fig. 15.

R-propranolol glucuronidation rates by dog UGT1As (upper panel) and human UGT1As (lower panel). See the legend to Fig. 3 for further details

Fig. 16.
Fig. 16.

S-propranolol glucuronidation rates by dog UGT1As (upper panel) and human UGT1As (lower panel). See the legend to Fig. 3 for further details.

Because this study focused on the dog (and human) UGTs of subfamily 1A, we tested their glucuronidation activity using compounds that were known to be primarily glucuronidated by one or more human UGTs of subfamily 1A. The activities of the human UGTs toward the selected compounds were in full agreement with the previously published studies that are cited here, confirming the analysis methods. The results with the dUGT1As, however, differed considerably from the results with human UGT1As.

We finally tested the recombinant dUGT1As with two substrates, testosterone and epitestosterone, that in humans are mainly glucuronidated by two different UGTs of subfamily 2B, UGT2B17 and UGT2B7, respectively (Sten et al., 2009). The results revealed very low dUGT1A activity toward either androgen, similar to the very low testosterone glucuronidation activity that was exhibited by some human UGT1As, which in turn was similar to what has been previously reported (see the supplemental materials, and Supplemental Fig. 2). Nevertheless, this low activity is an unlikely contributor to the very high testosterone glucuronidation activity that was exhibited by DLM.

Glucuronidation Activities in Dog and Human Liver and Intestine Microsomes.

In addition to testing the activities of dUGT1As and human UGT1As toward 14 compounds, we have also tested the activity of pooled microsomes from dog liver and intestine, as well as pooled human liver and intestine microsomes, for the glucuronidation of the same compounds (Figs. 17 and 18). The results show that in both species the glucuronidation rates, per milligram of microsomal protein, are mostly much higher in liver than in intestine microsomes, but not always. Examples for the latter are the rate of entacapone glucuronidation in DIM that is higher than in DLM, or the higher rate of E2-3-glucuronide formation in HIM than in HLM (Figs. 17 and 18). As for differences between DLM and HLM, perhaps the most striking are the differences in the glucuronidation rates of steroids at position 17, both the much higher rates of E2-17-glucuronide and testosterone glucuronide formation in DLM than in HLM (Fig. 17). It is also worth noting that ethinylestradiol-17-glucuronide was clearly detected in DLM, whereas practically no ethinylestradiol-17-glucuronide was detected in HLM (Fig. 17). A few more differences and similarities between the different microsomal preparations, such as the glucuronidation of bilirubin and entacapone, will be discussed herein.

Fig. 17.
Fig. 17.

Glucuronidation rates of the 14 test compounds used in this study by dog liver microsomes (DLM, upper panel) and human liver microsomes (HLM, lower panel). In the cases of estrogens, dexmedetomidine, and levomedetomidine, two different glucuronides were detected, and they are both indicated by marks on the top of the columns (see legends to Figs. 5 and 9).

Fig. 18.
Fig. 18.

Glucuronidation rates of the 14 test compounds used in this study by dog intestine microsomes (DIM, upper panel) and human intestine microsomes (HIM, lower panel). See the legend to Fig. 17 for further details.

Discussion

The beagle dog UGT enzymes of subfamily 1A were cloned and expressed as recombinant enzymes in baculovirus-infected insect cells. They were named according to Li and Wu (2007), rather than in full agreement with the exon 1s order in the large human ugt1a gene (Fig. 1). It is noteworthy that the degree of amino acid sequence similarity among the human UGT1As and dog dUGT1As, as revealed by phylogenetic tree analysis (Fig. 2), is lower than among some of the UGTs of the same species. This may be very important for substrate specificity, considering that even the high degree of sequence identity between the human UGTs 1A9 and 1A10 does not exclude large differences in their activities (Itäaho et al., 2010). Therefore, it may be wrong to assume that dog and human UGTs with similar names will have similar activities. On the other hand, the phylogenetic analysis provides an overview of the entire N-terminal halves (the domains encoded by exon 1 in the UGTs) of the sequence, and some specific residues within them may be much more important for a given activity than others. Because our current knowledge of the three-dimensional structure of the substrate-binding domain of the UGTs is still poor, the best way to examine the substrate specificity of the dog UGT1As and to compare it to the activities of the human UGT1As is to clone, express, and assay the activities of the enzymes.

Bilirubin glucuronidation in human is perhaps the most important activity of the UGTs, and it is (almost) specifically catalyzed by UGT1A1. The results suggest that in the dog it is dUGT1A1 that catalyzes this activity (Fig. 3). Interestingly, the rate of bilirubin glucuronidation by DLM was clearly lower than in HLM, in agreement with the lower bilirubin glucuronidation activity (corrected for relative expression level) of the recombinant dUGT1A1 in comparison with the human UGT1A1 (Fig. 3).

Racemic medetomidine is used also as a veterinary drug to treat dogs (Kuusela et al., 2000), and the results with DLM indicate that both medetomidine enantiomers can be glucuronidated in this tissue, as previously reported (Kaivosaari et al., 2008). Nevertheless, while human UGT1A4 was the most active enzyme in dexmedetomidine glucuronidation (Kaivosaari et al., 2008) (Fig. 9), none of the three dUGT1As that are closely related to it, dUGT1A2, dUGT1A3, and dUGT1A4, catalyze dexmedetomidine glucuronidation (Fig. 9). It is likely that they failed to catalyze this N-glucuronidation reaction because they have a His residue at the “catalytic His” position, position 39 of the protein precursor sequence (Li and Wu, 2007), while the human UGT1A4 has a Pro residue at this position (Kerdpin et al., 2009). It may be further suggested that both medetomidine enantiomers are glucuronidated in the dog by one or two UGTs of family 2, particularly if an equivalent of the human UGT2B10 is present there (Kaivosaari et al., 2008).

One of the large differences in activity between HLM and DLM was seen in entacapone glucuronidation rates, which are very high in HLM and low in DLM (Fig. 17). Interestingly, entacapone glucuronidation is almost the only activity of those tested in this study (the other one was ethinylestradiol-3-glucuronide formation) that was catalyzed at significantly higher rate in the dog intestine than in the liver (Figs. 18 versus 17). It is tempting to suggest that the relatively high entacapone glucuronidation rate in DIM, in comparison with DLM, indicates that dUGT1A11 is expressed in the dog intestine, but not in the dog liver. The latter suggestion turned out to be in agreement with a new study that was published while this article was under revision (Heikkinen et al., 2014). The line of reasoning or speculation on the issue of dUGT expression based on observed glucuronidation activities differences between DLM to DIM (Fig. 17), particularly the differences in the positions at which estrogens are conjugated, might be extended. Because most of the dUGT1As almost selectively catalyzed glucuronidation of the tested estrogens at the 3-OH (Figs. 4–6), the results may not only suggest that dUGT1A11 is an intestinal enzyme, but also that in the dog the UGTs of subfamily 2B and perhaps other subfamilies are significantly more important in the liver than in the intestine.

An interesting finding of this study was the identification of individual dUGTs, d1A8 and d1A10, that are able to catalyze the formation of linked diglucuronides of several estrogens, all three estrogens examined in this study. Both dUGT1A8 and dUGT1A10 produce only the E2-3-glucuronide when the substrate is E2, suggesting that E2 binds within their active site in a specific way that favors the production of E2-3-glucuronide (Fig. 5). However, when a much larger substrate binds, either E2-3-glucuronide or E2-17-glucuronide, it is not the estrogen backbone structure that determines the binding orientation in the enzyme’s substrate-binding site but the site at which the glucuronic acid moiety is attached to the estrogen backbone (Fig. 8). This suggests that to generate such linked diglucuronides, the 2′-hydroxyl of the first bound glucuronic acid should bind close to the “catalytic His” of the enzyme because this is the hydroxyl group previously found to be conjugated in such diglucuronides by the second glucuronic acid moiety (Murai et al., 2005; Argikar, 2012). The new finding—that the dUGT1A8 (or dUGT1A10) could bind either E2-3-glucuronide or E2-17-glucuronide in a “productive” way—gives rise to linked diglucuronides and may help future modeling of its binding site with bound E2-glucuronide in either orientation. This may be particularly interesting in the case of E2-17-glucuronide because, unlike E2-3-glucuronide, it is not a natural product of the enzyme.

It has been known for a long time that the human UGT1A1 can catalyze both mono- and diglucuronidation of bilirubin (see Zhou et al., 2010 for a kinetic analysis of the sequential reaction), and we also have noticed indications for this in dUGT1a1 (results not shown). Nevertheless, estrogens are different molecules than bilirubin—for example, in their rigidity and lack of pseudo-symmetry. In addition, unlike the estradiol diglucuronides described here, the bilirubin diglucuronides are not linked diglucuronides. Hence, there seems to be a significant difference between the human UGT1A1 and dUGT1A1 on the one hand, and dUGT1A8 and dUGT1A10 on the other, in the mechanism leading to the formation of diglucuronides.

Finally, in this study we have cloned and expressed the 10 dog UGTs of subfamily 1A, examined their glucuronidation activity, and compared it with the activities of the nine human UGTs of subfamily 1A that were expressed in the same system. In addition, we have examined the same activities in pooled liver and intestine microsomes from both dogs and humans. The results reveal many differences, alongside several similarities, at the levels of both individual enzymes and microsomes. The results provide many insights both into the structure and function of the UGTs and on the expression of individual UGTs in specific tissues. The results may also provide grounds for further consideration of interspecies differences and their practical values, even though we have not discussed this issue explicitly.

Acknowledgments

The authors thank Orion Pharma Corporation, Espoo, Finland, for the generous gifts of entacapone, dexmedetomidine and levomedetomidine, as well as Hannes Lohi, University of Helsinki, Finland, for a sample of beagle dog genomic DNA. We would also like to thank Dr. Peter I. Mackenzie, Flinders University, Adelaide, Australia, for verifying the correctness of the dog UGT1As sequences and for his useful comments about the way to name them. Thanks also to Dr. Linda Ahonen for her contribution to the LC-MS/MS analyses.

Authorship Contributions

Participated in research design: Finel, Troberg.

Conducted experiments: Järvinen, Troberg, Sneitz, Hagström, Muniz, Mosorin.

Performed data analysis: Troberg, Järvinen, Finel.

Wrote or contributed to the writing of the manuscript: Troberg, Järvinen, Finel.

Footnotes

    • Received May 29, 2014.
    • Accepted October 8, 2014.

  • J.T. and E.J. contributed equally to this study.

  • This study was supported by grants from the Academy of Finland [Grant 12600101] and the Sigrid Juselius Foundation [Grant 47033421].

  • dx.doi.org/10.1124/dmd.114.059303.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

Abbreviations

DMSO
dimethylsulfoxide
DIM
dog intestine microsomes
DLM
dog liver microsomes
E2
17-β-estradiol
epi-E2
17-α-estradiol
HIM
human intestine microsomes
HLM
human liver microsomes
HPLC
high-pressure liquid chromatography
LC-MS/MS
liquid chromatography with tandem mass spectrometry
4-MU
4-methylumbelliferone
PCA
perchloric acid
PCR
polymerase chain reaction
UDPGA
UDP-α-d-glucuronic acid
UGT
UDP-glucuronosyltransferase

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Recombinant Dog UGT1As

Johanna Troberg, Erkka Järvinen, Maria Muniz, Nina Sneitz, Johanna Mosorin, Marja Hagström and Moshe Finel
Drug Metabolism and Disposition January 1, 2015, 43 (1) 107-118; DOI: https://doi.org/10.1124/dmd.114.059303
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