Induction studies on the functional heterogeneity of rat liver UDP-glucuronosyltransferases

Differential induction with phenobarbital (PB) and 3-methylcholanthrene (3-MC) suggests at least two functionally distinct UDP glucuronosyltransferases (UDP-GT) which have different acceptor selectivities. One form is induced by 3-MC and preferentially conjugates group 1 acceptors, such as p-nitrophenol and 1-naphthol. Another UDP-GT is induced by PB and glucuronidates group 2 aglycones, morphine and chloramphenicol. To further study this functional heterogeneity, male Sprague-Dawley rats were pretreated with the following microsomal enzyme inducers: 7,8-benzoflavone (BF); benzo(a)pyrene (BP); 3-MC; 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); butylated hydroxyanisole (BHA); isosafrole; PB; pregnenolone-16α-carbonitrile (PCN); trans-stilbene oxide (TSO). The effect of induction on UDP-GT activity was determined with nine acceptors. Conjugation of group 1 aglycones, naphthol and p-nitrophenol, was increased by 3-MC (185 and 80%, respectively) whereas PB was ineffective. Conjugation of group 2 acceptors, morphine and chloramphenicol, was stimulated by PB (120 and 250%, respectively) while 3-MC had little effect. BP and TCDD enhanced glucuronidation of group 1 aglycones. ISF and TSO induced conjugation of both acceptor groups but were more effective for group 2. BF and BHA had negligible effects on UDP-GT activity. Since glucuronidation of valproic acid was increased only by PB and TSO treatment, this aglycone is probably a group 2 acceptor. Conjugation of digitoxigenin-monodigitoxoside (DIG) was stimulated by PB (200%) and PCN (1200%). PCN did not induce glucuronidation of group 1 acceptors but did have a slight effect on group 2 aglycones (130 and 40% for chloramphenicol and morphine, respectively). The 12-fold increase in DIG conjugation by PCN pretreated rats suggests that PCN may induce another group (form) of UDP-GT which preferentially glucuronidates DIG. Differential induction of UDP-GT activities within each group of acceptors indicates possible additional heterogeneity of the transferase.     

In vitro studies in microsomes from rat and human liver, kidney, and intestine suggest that perfluorooctanoic acid is not a substrate for microsomal UDP-glucuronosyltransferases

Perfluorooctanoic acid (PFOA) is a fluorinated fatty acid analogue used as a surfactant in the manufacture of fluoropolymers. Previous studies have indicated that PFOA was metabolically inert in mammals, but recent metabolism studies with related fluorochemicals suggested that PFOA might form a glucuronide conjugate. [(14)C(1)]-PFOA was incubated with male and female human and rat liver, kidney, and small intestine microsomes. Incubations were carried out in the presence of alamethicin and beta-saccharolactone to increase access of PFOA to the enzyme active site and to inhibit potential hydrolysis of PFOA-glucuronide by microsomal beta-glucuronidase, respectively. Although positive control experiments using p-nitrophenol demonstrated significant UDP-glucuronosyltransferase (UDPGT) activity in all of the tested microsomal preparations, no evidence for formation of a PFOA-glucuronide was obtained, either by high-sensitivity radiochromatography or by LC/MS. These data suggest that PFOA is not a substrate for human or rodent microsomal UDPGTs.     

Drug-metabolizing activity of human and rat liver,lung, kidney and intestine slices

1. Organ-specific biotransformation was studied in human and rat liver, lung, kidney and small intestine slices and compared on a protein basis, using four model substances. 2. Deethylation of lidocaine was highest in liver slices from both man and rat, followed by the small intestine. 3. Metabolism of testosterone was highest in liver slices, but a different overall metabolic pattern was found between the different organs. 4. Lung, kidney and intestine slices prepared from human and rat organs showed mainly an unknown metabolite of 7-ethoxycoumarin identified as 4-ethoxy-2-hydroxyphenyl propionic acid (EPPA). 5. The maximal metabolism of 7-ethoxycoumarin in slices was equal with in vivo V_max in the rat. 6. Phase II metabolism of 7-hydroxycoumarin in kidney and intestinal slices was about 60% of the activity in liver slices. 7. In conclusion, organs other than the liver show a surprisingly high drug-metabolizing activity. Thus, the use of precision-cut slices of a combination of drug metabolizing organs in an in vitro test system from both animal and human origin is required for a proper systematic prediction of drug metabolism in man.     

Drug-metabolizing activity of human and rat liver,lung, kidney and intestine slices

1. Organ-specific biotransformation was studied in human and rat liver, lung, kidney and small intestine slices and compared on a protein basis, using four model substances. 2. Deethylation of lidocaine was highest in liver slices from both man and rat, followed by the small intestine. 3. Metabolism of testosterone was highest in liver slices, but a different overall metabolic pattern was found between the different organs. 4. Lung, kidney and intestine slices prepared from human and rat organs showed mainly an unknown metabolite of 7-ethoxycoumarin identified as 4-ethoxy-2-hydroxyphenyl propionic acid (EPPA). 5. The maximal metabolism of 7-ethoxycoumarin in slices was equal with in vivo V(max) in the rat. 6. Phase II metabolism of 7-hydroxycoumarin in kidney and intestinal slices was about 60% of the activity in liver slices. 7. In conclusion, organs other than the liver show a surprisingly high drug-metabolizing activity. Thus, the use of precision-cut slices of a combination of drug metabolizing organs in an in vitro test system from both animal and human origin is required for a proper systematic prediction of drug metabolism in man.     

Determination of the kinetics of rat UDP-glucuronosyltransferases (UGTs) in liver and intestine using HPLC

Uridinediphosphoglucuronosyl transferases (UGTs) are a group of membrane bound proteins which catalyze the transfer of glucuronic acid from UDP-glucuronic acid to a wide variety of xenobiotics and drug molecules enabling them to be eliminated. The major UGT isoforms found in the rat are 1A1, 1A6, 2B1 and 2B12. Conventional methods for the assay of glucuronides (GLs) include TLC, extraction and colorimetry or quantification of the aglycone, liberated after hydrolyzing the GL with beta-glucuronidase. However these techniques cannot distinguish between isomeric GLs or GLs of multiple acceptor site substrates. Therefore the purpose of this study was to develop simple and sensitive HPLC methods for the direct and simultaneous analysis of the GL(s) and their aglycones without the drawbacks of the conventional methods. The three classical substrates we chose were 4-methylumbelliferone (4MU), testosterone (TES) and 8-hydroxyquinoline (8HOQ) representing UGT isoforms 1A6, 2B1 and 2B12 of the rat family, respectively. Here we report the validated HPLC conditions, for the detection and separation of 4-methylumbelliferone glucuronide (4MUG), testosterone glucuronide (TESG) and 8-hydroxyquinoline glucuronide (8HOQG) and their aglycones in incubation media containing male Sprague-Dawley rat liver and intestinal microsomal preparations. The separations were achieved on a Zorbax SB-CN column (150 x 4.6 mm, 5 micron). The analysis time for the separation of TES, 8HOQ and 4MU and their glucuronides were 17, 12 and 30 min, respectively. The methods showed excellent linearity (r2 > 0.99) over the concentration ranges tested (0.25-5.0 nmoles of TESG; 0.125-18.75 nmoles of 8HOQG and 0.125-12.5 nmoles of 4MUG), good precision and accuracy (RSD<2.5%). Inter-day variability studies (n = 3) showed no significant difference between the regression lines obtained on the three days. Recoveries were good ( > 90%) at all three points (low, mid-point, high) of the standard curve. The limits of detection were 0.125, 0.1 and 0.1 nmole for TESG, 8HOQG and 4MUG. respectively. The above methods were used to estimate kinetic parameters such as Vmax and Km for the GLs of the three substrates in both liver and intestinal tissue preparations and the values were comparable with previously reported results. UGT2B1 was found primarily in the liver while UGTs 1A6 and 2B12 were present in comparable amounts in both tissues.     

Drug transport in intestine,liver and kidney

Drug transport in intestine, liver and kidney is similar, because in each case transport occurs across a barrier of epithelial cells. However, the physiological conditions differ in each organ: intestinal drug absorption is largely influenced by physicochemical conditions in the intestinal lumen; actual transport across the epithelial barrier occurs mainly by diffusion; carrier-mediated transport plays a subordinate role. In contrast, hepatic uptake is mediated by specific carriers, which transport a wide variety of drugs into the liver cell and then release them either into bile, or back into the portal blood. It is unclear how many carrier systems are involved, how they are organized in the liver cell membrane, and to what extent their substrate specificities overlap. Renal secretion and reabsorption of drugs is mediated by highly active carrier systems for cations and anions. Their cooperative action results in either active reabsorption or active secretion of drugs.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

Methylation of captopril in human liver,kidney and intestine

1. The methylation of captopril was studied in the microsomal fraction from 20 human liver, 12 kidney, and 14 intestinal rnucosa specimens.

2. The hepatic methyltransferase activity (mean ± SD) was 477 ± 204 pmol/min per mg. Renal and intestinal methyltransferase activities were 3 and 8 times lower, respectively, than hepatic activity.

3. The kinetics of methyltransferase with captopril as substrate were studied in four specimens of liver, kidney and intestine. The maximum velocities of reaction (mean ± SD; pmol/min per?mg) were 697 ± 219 (liver), 456 ± 120 (renal cortex), 264 ± 77 (renal medulla) and 101 ± 28 (ileum mucosa). K_m values (mean ± SD; mM) were 5.2±2.3 (liver) 4.3±1.7 (renal cortex) 4.1±1.5 (renal medulla) and 5.3 ± 2.0 mM (ileum mucosa). V_max is subjected to a marked tissue dependence whereas K_m is similar in all tissues.

4. Liver is the primary site of captopril methylation whereas the intestine plays only a minor role. Kidney may contribute substantially to the hepatic methylation of captopril.     

Human and rat liver UDP-glucuronosyltransferases are targets of ketoprofen acylglucuronide.

Acylglucuronides formed from carboxylic acids by UDP-glucuronosyltransferases (UGTs) are electrophilic metabolites able to covalently bind proteins. In this study, we demonstrate the reactivity of the acylglucuronide from the nonsteroidal anti-inflammatory drug, ketoprofen, toward human and rat liver UGTs. Ketoprofen acylglucuronide irreversibly inhibited the glucuronidation of 1-naphthol and 2-naphthol catalyzed by human liver microsomes or by the recombinant rat liver isoform, UGT2B1, which is the main isoform involved in the glucuronidation of the drug. A decrease of about 35% in the glucuronidation of 2-naphthol was observed when ketoprofen acylglucuronide was produced in situ in cultured V79 cells expressing UGT2B1. Inhibition was always associated with the formation of microsomal protein-ketoprofen adducts. The presence of these covalent adducts within the endoplasmic reticulum of cells expressing UGT2B1 was demonstrated following addition of ketoprofen to culture medium by immunofluorescence microscopy with antiketoprofen antibodies. Immunoblots of liver microsomes incubated with ketoprofen acylglucuronide and probed with antiketoprofen antibodies revealed the presence of several protein adducts; among those was a major immunoreactive protein at 56 kDa, in the range of the apparent molecular mass of UGTs. The adduct formation partially prevented the photoincorporation of the UDP-glucuronic acid (UDP-GlcUA) analog, [beta-32P]5N3UDP-GlcUA, on the UGTs, suggesting that ketoprofen glucuronide covalently reacted with the UDP-GlcUA binding domain. Finally, UGT purification from rat liver microsomes incubated with ketoprofen glucuronide led to the isolation of UGT adducts recognized by both anti-UGT and antiketoprofen antibodies, providing strong evidence that UGTs are targets of this metabolite.     

Functional heterogeneity of UDP-glucuronosyltransferases in different membranes of rat liver

Glucuronidation of molecules of very different shape suggests the existence of several enzymes with different acceptor specificities. In the endoplasmic reticulum, two kinds of activities have already been characterized. The late fetal activity (1), which is enhanced by 3-methylcholanthrene (2) is able to conjugate planar molecules or group I substrates (3). The neonatal activity (1), inducible by phenobarbital (2), shows specificity towards bulky aglycones or group II substrates (3). Although the endoplasmic reticulum of hepatocytes is known as the major site of xenobiotic glucuronoconjugation, we have recently detected the presence of UDP-glucuronosyltransferase (EC 2.4.1.17) with 4-nitrophenol as the substrate, in the plasma membrane fraction of rabbit liver, which can be induced by phenobarbital (4). Such an activity has also been discovered in Golgi apparatus and in nuclear membranes (5, 6). In order to establish if the enzyme heterogeneity according to the type of substrate used also exists in the different subcellular organelles, we have investigated UDP-glucuronosyltransferase activity towards 10 aglycones In plasma membranes (PM), Golgi apparatus, nuclear envelope and rough or smooth endoplasmic reticulum (RER, SER) of rat liver.     

Structural and functional studies of UDP-glucuronosyltransferases

UDP-Glucuronosyltransferases (UGTs) are glycoproteins localized in the endoplasmic reticulum (ER) which catalyze the conjugation of a broad variety of lipophilic aglycon substrates with glucuronic acid using UDP-glucuronic acid (UDP-GIcUA) as the sugar donor. Glucuronidation is a major factor in the elimination of lipophilic compounds from the body. In this review, current information on the substrate specificities of UGT1A and 2B family isoforms is discussed. Recent findings with regard to UGT structure and topology are presented, including a dynamic topological model of UGTs in the ER. Evidence from experiments on UGT interactions with inhibitors directed at specific amino acids, photoaffinity labeling, and analysis of amino acid alignments suggest that UDP-GIcUA interacts with residues in both the N- and C-terminal domains, whereas aglycon binding sites are localized in the N-terminal domain. The amino acids identified so far as crucial for substrate binding and catalysis are arginine, lysine, histidine, proline, and residues containing carboxylic acid. Site-directed mutagenesis experiments are critical for unambiguous identification of the active-site architecture.     

Isolation and characterization of metabolites of centpropazine in rat liver,intestine, and red blood cell homogenates

The potential sites for metabolism of centpropazine (CPZ) (an antidepressant) were evaluated in male Sprague-Dawley rats. The isolation and identification of the major metabolites formed in the presence of rat liver S9 fraction, intestine, and red blood cells under aerobic conditions were performed using high-performance liquid chromatography and electrospray ionization mass spectrometry. CPZ was found to be extensively metabolized to seven possible metabolites by liver S9 fraction in the presence of a nicotinamide adenine dinucleotide phosphate generating system at 37 degrees C. Both intestinal wall and red blood cells were also found to metabolize the compound. This metabolite structure was confirmed by comparison with that of its synthetic standard. The drug was stable in intestinal contents. On the basis of our finding, we propose the in vitro metabolic pathways for CPZ.     

Methylation of captopril in human liver, kidney and intestine.

1. The methylation of captopril was studied in the microsomal fraction from 20 human liver, 12 kidney, and 14 intestinal mucosa specimens. 2. The hepatic methyltransferase activity (mean +/- SD) was 477 +/- 204 pmol/min per mg. Renal and intestinal methyltransferase activities were 3 and 8 times lower, respectively, than hepatic activity. 3. The kinetics of methyltransferase with captopril as substrate were studied in four specimens of liver, kidney and intestine. The maximum velocities of reaction (mean +/- SD; pmol/min per mg) were 697 +/- 219 (liver), 456 +/- 120 (renal cortex), 264 +/- 77 (renal medulla) and 101 +/- 28 (ileum mucosa). Km values (mean +/- SD; mM) were 5.2 +/- 2.3 (liver) 4.3 +/- 1.7 (renal cortex) 4.1 +/- 1.5 (renal medulla) and 5.3 +/- 2.0 mM (ileum mucosa). Vmax is subjected to a marked tissue dependence whereas Km is similar in all tissues. 4. Liver is the primary site of captopril methylation whereas the intestine plays only a minor role. Kidney may contribute substantially to the hepatic methylation of captopril.     

Inhibition of UDP-glucuronosyltransferases in rat liver microsomes by natural mutagens and carcinogens

Eight toxic compounds of natural origin present in the human diet or used as drugs were tested as inhibitors of UDP-glucuronosyltransferase (UGT) activity in rat liver microsomes with 1-naphthol (1-NA), phenolphthalein (PPh) and 4-nitrophenol (4-NP) as substrates. Strong inhibitory effects were observed with tannic acid (tannin) and the antifungal drug griseofulvin (GF): at a concentration of 1 mM, the two compounds completely suppressed the glucuronidation of 1-NA and PPh, respectively. A concentration of 0.1 mM still proved to be highly inhibitory, and even at a concentration as low as 50 microM, tannin produced nearly 50% inhibition of 1-NA conjugation. The UGT isoforms converting 4-NP were less sensitive to the tested compounds (with the exception of GF). Kinetic studies with tannin revealed an uncompetitive type of inhibition toward 1-NA, with an apparent Ki value of 20 microM. The inhibition by GF was non-competitive with respect to PPh and was of a mixed type toward UDP-glucuronic acid, with apparent Ki values of 40 microM and 30 microM, respectively. Tannin and GF did not act as substrates for rat microsomal UGT.     

Prediction of whole-body metabolic clearance of drugs through the combined use of slices from rat liver,lung, kidney,small intestine and colon

1.?The aim was to investigate whether precision-cut rat tissue slices could be used to predict metabolic drug clearance in vivo. To obtain a complete picture, slices not only from liver, but also from lung, kidney, small intestine and colon were included.

2.?The metabolic clearances of 7-ethoxycoumarin, 7-hydroxycoumarin, testosterone, methyltestosterone and warfarin were determined by measuring the disappearance of these compounds during incubation with slices prepared from liver, lung, kidney, small intestine and colon.

3.?The total in vitro metabolic clearance was determined by adding the individual in vitro organ clearances from the slices. Prediction based on the in vitro clearance was within an order of magnitude to the corresponding in vivo values. Interestingly, the relative contribution of extrahepatic metabolic clearance of the studied compounds to total clearance was remarkably high, ranging from 35 to 72% of the total metabolic clearance.

4.?It is concluded that the model of multi-organ precision-cut slices is a useful in vitro tool for prediction of in vivo metabolic clearance. In addition, it provides information about the relative contribution of the liver, lung, kidney, small intestine and colon to the total metabolic clearance.     

Prediction of whole-body metabolic clearance of drugs through the combined use of slices from rat liver, lung, kidney, small intestine and colon

1: The aim was to investigate whether precision-cut rat tissue slices could be used to predict metabolic drug clearance in vivo. To obtain a complete picture, slices not only from liver, but also from lung, kidney, small intestine and colon were included. 2: The metabolic clearances of 7-ethoxycoumarin, 7-hydroxycoumarin, testosterone, methyltestosterone and warfarin were determined by measuring the disappearance of these compounds during incubation with slices prepared from liver, lung, kidney, small intestine and colon. 3: The total in vitro metabolic clearance was determined by adding the individual in vitro organ clearances from the slices. Prediction based on the in vitro clearance was within an order of magnitude to the corresponding in vivo values. Interestingly, the relative contribution of extrahepatic metabolic clearance of the studied compounds to total clearance was remarkably high, ranging from 35 to 72% of the total metabolic clearance. 4: It is concluded that the model of multi-organ precision-cut slices is a useful in vitro tool for prediction of in vivo metabolic clearance. In addition, it provides information about the relative contribution of the liver, lung, kidney, small intestine and colon to the total metabolic clearance.     

The distribution of UDP-glucuronosyltransferases in rat liver parenchymal and nonparenchymal cells.

Activities for the glucuronidation of 1-naphthol, morphine and bilirubin as well as for the sulfation of 2-naphthol have been determined in homogenates of parenchymal, Kupffer and endothelial cells isolated from livers of untreated and Aroclor 1254-pretreated rats. In addition, Western blot analyses using different polyclonal antibodies against UDP-glucuronosyltransferases (UDP-GTs) were performed with similar preparations. All enzymes under investigation were expressed at high levels in liver parenchymal cells. The constitutive expression and inducibility of UDP-GT isozyme(s) for 1-naphthol glucuronidation was also clearly demonstrated in Kupffer and endothelial cells. Furthermore, the presence of other UDP-GT isozymes was detected in preparations from these cells. No significant sulfation of 2-naphthol was detectable in Kupffer and endothelial cell homogenates. While the glucuronidation of 1-naphthol and morphine was significantly induced in all cell types by Aroclor 1254-pretreatment of the animals, the glucuronidation of bilirubin and the sulfation of 2-naphthol remained unchanged. Since the specific activity of conjugation reactions is much lower in liver nonparenchymal cells than in liver parenchymal cells, and nonparenchymal cells contribute only about 6% to the total liver protein, protection of the cells themselves rather than contribution to the overall metabolism of xenobiotics seems to be the significant role of these xenobiotic-metabolizing enzymes in the sinusoidal lining cells.     

Immunoquantitation of FMO1 in human liver, kidney, and intestine.

To determine the level of FMO1 protein present in human liver tissues, a monospecific antibody was prepared and a sensitive Western blotting procedure with enhanced chemiluminescence detection was developed. Human FMO1, purified from insect cells expressing the recombinant protein, was used as a protein standard for absolute quantification. The average concentrations of FMO1 in microsomes prepared from human liver, kidney, intestine, and fetal liver were found to be <1, 47 +/- 9, 2.9 +/- 1.9, and 14.4 +/- 3.5 pmol/mg, respectively. Quantitation in intestinal microsomes was complicated by variable degrees of proteolytic degradation of FMO1, not seen in microsomes prepared from liver or kidney. Recombinant human FMO1 and detergent-solubilized human duodenal microsomes both metabolized p-tolyl methyl sulfide stereoselectively to the (R)-sulfoxide, indicating the expression of functional FMO1 in human intestine. The relatively high levels of immunoquantifiable FMO1 in human kidney and fetal liver complement our previous catalytic studies in these tissues, which also demonstrated preferential (R)-p-tolyl methyl sulfoxide formation. These data demonstrate a profound ontogenic change in expression of hepatic FMO1 in humans, such that in adult life FMO1 is exclusively an extrahepatic drug-metabolizing enzyme. The marked expression levels of FMO1 found in human kidney coupled to the high catalytic activity of this isoform toward a diverse array of sulfides and tertiary amines suggest the possibility that human renal FMO1 is a significant contributor to the metabolic clearance of drugs and other xenobiotics bearing these functionalities.     

Identification of UDP-glucuronosyltransferases responsible for the glucuronidation of darexaban, an oral factor Xa inhibitor, in human liver and intestine

Darexaban maleate is a novel oral direct factor Xa inhibitor, which is under development for the prevention of venous thromboembolism. Darexaban glucuronide was the major component in plasma after oral administration of darexaban to humans and is the pharmacologically active metabolite. In this study, we identified UDP-glucuronosyltransferases (UGTs) responsible for darexaban glucuronidation in human liver microsomes (HLM) and human intestinal microsomes (HIM). In HLM, the K(m) value for darexaban glucuronidation was >250 μM. In HIM, the reaction followed substrate inhibition kinetics, with a K(m) value of 27.3 μM. Among recombinant human UGTs, UGT1A9 showed the highest intrinsic clearance for darexaban glucuronidation, followed by UGT1A8, -1A10, and -1A7. All other UGT isoforms were inactive toward darexaban. The K(m) value of recombinant UGT1A10 for darexaban glucuronidation (34.2 μM) was comparable to that of HIM. Inhibition studies using typical UGT substrates suggested that darexaban glucuronidation in both HLM and HIM was mainly catalyzed by UGT1A8, -1A9, and -1A10. Fatty acid-free bovine serum albumin (2%) decreased the unbound K(m) for darexaban glucuronidation from 216 to 17.6 μM in HLM and from 35.5 to 18.3 μM in recombinant UGT1A9. Recent studies indicated that the mRNA expression level of UGT1A9 is extremely high among UGT1A7, -1A8, -1A9, and -1A10 in human liver, whereas that of UGT1A10 is highest in the intestine. Thus, the present results strongly suggest that darexaban glucuronidation is mainly catalyzed by UGT1A9 and UGT1A10 in human liver and intestine, respectively. In addition, UGT1A7, -1A8, and -1A9 play a minor role in human intestine.