Ryoichi Fujiwara

Expression of UGT1A and UGT2B mRNA in Human Normal Tissues and Various Cell Lines

UDP-glucuronosyltransferases (UGTs) are major phase II drug metabolism enzymes that catalyze the glucuronidation of numerous endogenous and exogenous compounds. UGTs are divided into two families, UGT1 and UGT2, based on evolutionary divergence and homology. Nine UGT1A and seven UGT2B functional isoforms have been identified in humans. Glucuronidation occurs mainly in liver but also in various extrahepatic tissues, possibly affecting the pharmacokinetics. In the present study, we comprehensively determined the expression of all functional UGT1A and UGT2B isoforms in normal human tissues including liver, lung, stomach, small intestine, colon, kidney, bladder, adrenal gland, breast, ovary, uterus, and testis by semiquantitative reverse transcription-polymerase chain reaction. In addition, the expressions of these UGTs mRNA in 15 kinds of human tissue-derived cell lines were also analyzed. Many UGT isoforms were abundantly expressed in the liver, gastrointestinal tract, and kidney, supporting previous studies. Interestingly, we found that all UGTs except UGT2B17 were expressed in bladder. In steroid-related tissues, UGTs were expressed in tissue- and isoform-specific manners. Expression profiles in human tissue-derived cell lines were not necessarily consistent with those in corresponding normal tissues. Different expression profiles were observed in distinct cell lines derived from the same organ. The information presented here will be helpful for understanding the glucuronidation in various tissues and for choosing appropriate cell lines for in vitro studies.

Quantitative Analysis of UDP-Glucuronosyltransferase (UGT) 1A and UGT2B Expression Levels in Human Livers

UDP-glucuronosyltransferases (UGTs) catalyze glucuronidation of a variety of xenobiotics and endobiotics. UGTs are divided into two families, UGT1 and UGT2. The purpose of this study was to estimate the absolute expression levels of each UGT isoform in human liver and to evaluate the interindividual variability. Real-time reverse transcriptase-polymerase chain reaction analysis was performed to determine the copy numbers of nine functional UGT1A isoforms and seven UGT2B isoforms. We noticed that not only primers but also templates as a standard for quantification should prudently be selected. Once we established appropriate conditions, the mRNA levels of each UGT isoform in 25 individual human livers were determined. UGT1A1 (0.9–138.5), UGT1A3 (0.1–66.6), UGT1A4 (0.1–143.3), UGT1A6 (1.0–70.4), UGT1A9 (0.3–132.4), UGT2B4 (0.3–615.0), UGT2B7 (0.2–97.4), UGT2B10 (0.7–253.2), UGT2B15 (0.3–107.8), and UGT2B17 (0.5–157.1) were substantially expressed (×104 copy/μg RNA) with large interindividual variability. Abundant isoforms were UGT2B4 and UGT2B10, followed by UGT1A1, UGT2B15, and UGT1A6. The sum of the UGT2B mRNA levels was higher than that of UGT1A mRNA levels. It is interesting to note that the mRNA levels normalized with glyceraldehyde-3-phosphate dehydrogenase mRNA for almost UGT isoforms that are substantially expressed in liver showed significant correlations to each other. Western blot analysis was performed using antibodies specific for UGT1A1, UGT1A4, UGT1A6, or UGT2B7. Correlation between the protein and mRNA levels was observed in only UGT1A1 (r = 0.488; p < 0.01). In conclusion, this study comprehensively determined the absolute values of mRNA expression of each UGT isoform in human livers and found considerable interindividual variability.

Developmental hyperbilirubinemia and CNS toxicity in mice humanized with the UDP glucuronosyltransferase 1 (UGT1) locus

High levels of unconjugated bilirubin (UCB) in newborn children is associated with a reduction in hepatic UDP glucuronosyltransferase (UGT) 1A1 activity that can lead to CNS toxicity, brain damage, and even death. Little is known regarding those events that lead to UCB accumulation in brain tissue, and therefore, we sought to duplicate this condition in mice. The human UGT1 locus, encoding all 9-UGT1A genes including UGT1A1, was expressed in Ugt1−/− mice. Because the most common clinical condition associated with jaundice in adults is Gilbert’s syndrome, which is characterized by an allelic polymorphism in the UGT1A1 promoter, hyperbilirubinemia was monitored in humanized UGT1 mice that expressed either the Gilbert’s UGT1A1*28 allele [Tg(UGT1A1*28)Ugt1−/− mice] or the normal UGT1A1*1 allele [Tg(UGT1A1*1)Ugt1−/− mice]. Adult Tg(UGT1A1*28)Ugt1−/− mice expressed elevated levels of total bilirubin (TB) compared with Tg(UGT1A1*1)Ugt1−/− mice, confirming that the promoter polymorphism associated with the UGT1A1*28 allele contributes to hyperbilirubinemia in mice. However, TB accumulated to near toxic levels during neonatal development, a finding that is independent of the Gilbert’s UGT1A1*28 promoter polymorphism. Whereas serum TB levels eventually returned to adult levels, TB clearance in neonatal mice was not associated with hepatic UGT1A1 expression. In ∼10% of the humanized UGT1 mice, peak TB levels culminated in seizures followed by death. UCB deposition in brain tissue and the ensuing seizures were associated with developmental milestones and can be prevented by enhancing regulation of the UGT1A1 gene in neonatal mice.

Toxicological evaluation of acyl glucuronides of nonsteroidal anti-inflammatory drugs using human embryonic kidney 293 cells stably expressing human UDP-glucuronosyltransferase and human hepatocytes.

The chemical reactivity of acyl glucuronide (AG) has been thought to be associated with the toxic properties of drugs containing carboxylic acid moieties, but there has been no direct evidence that AG formation was related to the toxicity. In the present study, the cytotoxicity and genotoxicity of AGs were investigated. Human embryonic kidney (HEK) 293 cells stably expressing UDP-glucuronosyltransferase (UGT) 1A3 (HEK/UGT1A3) were constructed to assess the cytotoxicity of AGs, and HEK/UGT1A4 cells were also used as a negative reference. After exposure to nonsteroidal anti-inflammatory drugs (NSAIDs) such as naproxen (1 mM), diclofenac (0.1 mM), ketoprofen (1 mM), or ibuprofen (1 mM) for 24 h, HEK/UGT1A3 cells produced AG in a time-dependent manner. However, HEK/UGT1A4 cells hardly produced AG. The cytotoxicity of HEK/UGT1A3 cells was not increased compared with that of HEK/UGT1A4 cells. In addition, the AG formed in NSAID-treated human hepatocytes was decreased from one-third to one-ninth by treatment with (−)-borneol, an inhibitor of acyl glucuronidation, but the cytotoxicity was increased. These results indicated that AG formation reflected the detoxification process in human hepatocytes. Furthermore, the possibility of genotoxicity from the AG formed in NSAID-treated HEK/UGT cells was investigated by the comet assay, and DNA damage was not detected in any HEK/UGT cell lines. In conclusion, the in vitro cytotoxic and genotoxic effects of the AGs of NSAIDs were investigated and AG was not found to be a causal factor in the toxicity.

Stereoselective Glucuronidation of 5-(4′-Hydroxyphenyl)-5-phenylhydantoin by Human UDP-Glucuronosyltransferase (UGT) 1A1, UGT1A9, and UGT2B15: Effects of UGT-UGT Interactions

5-(4′-Hydroxyphenyl)-5-phenylhydantoin (4′-HPPH), a major metabolite of phenytoin in human, is exclusively metabolized to a glucuronide. 4′-HPPH has a chiral center. (S)-4′-HPPH is a predominant form produced from phenytoin in humans, and (R)-4′-HPPH is an extremely toxic form with respect to gingival hyperplasia. In the present study, we investigated stereoselective 4′-HPPH O-glucuronide formation in human liver microsomes. Human liver microsomes predominantly formed (S)-4′-HPPH O-glucuronide rather than (R)-4′-HPPH O-glucuronide from racemic 4′-HPPH. Among human UDP-glucuronosyltransferase (UGT) enzymes, UGT1A1, UGT1A9, and UGT2B15 showed 4′-HPPH O-glucuronide formation. Interestingly, UGT1A1 stereoselectively formed (R)-4′-HPPH O-glucuronide, whereas UGT1A9 and UGT2B15 stereoselectively formed (S)-4′-HPPH O-glucuronide from racemic 4′-HPPH. By using UGT1A double-expression systems in HEK293 cells that we previously established, the effects of UGT-UGT interactions on 4′-HPPH O-glucuronide formation were investigated. It was demonstrated that coexpression of UGT1A4 increased the Vmax values of (S)- and (R)-4′-HPPH O-glucuronide formation catalyzed by UGT1A1 but decreased the Vmax values of (S)- and (R)-4′-HPPH O-glucuronide formation catalyzed by UGT1A9. Coexpression of UGT1A6 increased the S50 values and decreased the Vmax values of (S)- and (R)-4′-HPPH glucuronide formation catalyzed by UGT1A1 and UGT1A9. However, the interaction did not alter the stereoselectivity. In conclusion, we found that 4′-HPPH O-glucuronide formation in human liver microsomes is catalyzed by UGT1A1, UGT1A9, and UGT2B15 in a stereoselective manner, being modulated by interaction with other UGT1A isoforms.

Importance of UDP-glucuronosyltransferase 1A1 expression in skin and its induction by UVB in neonatal hyperbilirubinemia.

UDP-glucuronosyltransferase (UGT) 1A1 is the sole enzyme that can metabolize bilirubin. Human infants physiologically develop hyperbilirubinemia as the result of inadequate expression of UGT1A1 in the liver. Although phototherapy using blue light is effective in preventing jaundice, sunlight has also been suggested, but without conclusive evidence, to reduce serum bilirubin levels. We investigated the mRNA expression pattern of human UGT1A1 in human skin, human skin keratinocyte (HaCaT) cells, and skin of humanized UGT1 mice. The effects of UVB irradiation on the expression of UGT1A1 in the HaCaT cells were also examined. Multiple UGT1A isoforms, including UGT1A1, were expressed in human skin and HaCaT cells. When HaCaT cells were treated with UVB-exposed tryptophan, UGT1A1 mRNA and activity were significantly induced. Treatment of the HaCaT cells with 6-formylindolo[3,2-b]carbazole, which is one of the tryptophan derivatives formed by UVB, resulted in an induction of UGT1A1 mRNA and activity. In neonates, the expression of UGT1A1 was greater in the skin; in adults, UGT1A1 was expressed mainly in the liver. Treatment of humanized UGT1 mice with UVB resulted in a reduction of serum bilirubin levels, along with increased UGT1A1 expression and activity in the skin. Our data revealed a protective role of UGT1A1 expressed in the skin against neonatal hyperbilirubinemia. Sunlight, a natural and free source of light, makes it possible to treat neonatal jaundice while allowing mothers to breast-feed neonates.

Role of extrahepatic UDP-glucuronosyltransferase 1A1: Advances in understanding breast milk-induced neonatal hyperbilirubinemia.

Newborns commonly develop physiological hyperbilirubinemia (also known as jaundice). With increased bilirubin levels being observed in breast-fed infants, breast-feeding has been recognized as a contributing factor for the development of neonatal hyperbilirubinemia. Bilirubin undergoes selective metabolism by UDP-glucuronosyltransferase (UGT) 1A1 and becomes a water soluble glucuronide. Although several factors such as gestational age, dehydration and weight loss, and increased enterohepatic circulation have been associated with breast milk-induced jaundice (BMJ), deficiency in UGT1A1 expression is a known cause of BMJ. It is currently believed that unconjugated bilirubin is metabolized mainly in the liver. However, recent findings support the concept that extrahepatic tissues, such as small intestine and skin, contribute to bilirubin glucuronidation during the neonatal period. We will review the recent advances made towards understanding biological and molecular events impacting BMJ, especially regarding the role of extrahepatic UGT1A1 expression.

Glucuronidation of drugs in humanized UDP-glucuronosyltransferase 1 mice: Similarity with glucuronidation in human liver microsomes.

Uridine 5′‐diphosphate‐glucuronosyltransferases (UGTs) are phase II drug‐metabolizing enzymes that catalyze glucuronidation of various endogenous and exogenous substrates. Among 19 functional human UGTs, UGT1A family enzymes largely contribute to the metabolism of clinically used drugs. While the UGT1A locus is conserved in mammals such as humans, mice, and rats, species differences in drug glucuronidation have been reported. Recently, humanized UGT1 mice in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus (hUGT1 mice) have been developed. To evaluate the usefulness of hUGT1 mice to predict human glucuronidation of drugs, UGT activities, and inhibitory effects on UGTs were examined in liver microsomes of hUGT1 mice as well as in those of wild‐type mice and humans. Furosemide acyl‐glucuronidation was sigmoidal and best fitted to the Hill equation in hUGT1 mice and human liver microsomes, while it was fitted to the substrate inhibition equation in mouse liver microsomes. Kinetic parameters of furosemide glucuronidation were very similar between hUGT1 mice and human liver microsomes. The kinetics of S‐naproxen acyl‐glucuronidation and inhibitory effects of compounds on furosemide glucuronidation in hUGT1 liver microsomes were also slightly, but similar to those in human liver microsomes, rather than in wild‐type mice. While wild‐type mice lack imipramine and trifluoperazine N‐glucuronidation potential, hUGT1 mice showed comparable N‐glucuronidation activity to that of humans. Our data indicate that hUGT1 mice are promising tools to predict not only in vivo human drug glucuronidation but also potential drug‐drug interactions.

Impact of fatty acids on human UDP-glucuronosyltransferase 1A1 activity and its expression in neonatal hyperbilirubinemia

While breast milk has been known as a cause of neonatal hyperbilirubinemia, the underlying mechanism of breast milk-induced jaundice has not been clarified. Here, the impact of fatty acids on human UDP-glucuronosyltransferase (UGT) 1A1 – the sole enzyme that can metabolize bilirubin – were examined. Oleic acid, linoleic acid, and docosahexaenoic acid (DHA) strongly inhibited UGT1A1 activity. Forty-eight hours after a treatment with a lower concentration of DHA (10 mg/kg), total bilirubin significantly increased in neonatal hUGT1 mice, which are human neonatal jaundice models. In contrast, treatments with higher concentrations of fatty acids (0.1–10 g/kg) resulted in a decrease in serum bilirubin in hUGT1 mice. It was further demonstrated that the treatment with higher concentrations of fatty acids induced UGT1A1, possibly by activation of peroxisome proliferator-activated receptors. Our data indicates that activation of peroxisome proliferator-activated receptors would increase UGT1A1 expression, resulting in reduction of serum bilirubin levels in human infants.

Glucose induces intestinal human UDP-glucuronosyltransferase (UGT) 1A1 to prevent neonatal hyperbilirubinemia

Inadequate calorie intake or starvation has been suggested as a cause of neonatal jaundice, which can further cause permanent brain damage, kernicterus. This study experimentally investigated whether additional glucose treatments induce the bilirubin-metabolizing enzyme – UDP-glucuronosyltransferase (UGT) 1A1 – to prevent the onset of neonatal hyperbilirubinemia. Neonatal humanized UGT1 (hUGT1) mice physiologically develop jaundice. In this study, UGT1A1 expression levels were determined in the liver and small intestine of neonatal hUGT1 mice that were orally treated with glucose. In the hUGT1 mice, glucose induced UGT1A1 in the small intestine, while it did not affect the expression of UGT1A1 in the liver. UGT1A1 was also induced in the human intestinal Caco-2 cells when the cells were cultured in the presence of glucose. Luciferase assays demonstrated that not only the proximal region (-1300/-7) of the UGT1A1 promoter, but also distal region (-6500/-4050) were responsible for the induction of UGT1A1 in the intestinal cells. Adequate calorie intake would lead to the sufficient expression of UGT1A1 in the small intestine to reduce serum bilirubin levels. Supplemental treatment of newborns with glucose solution can be a convenient and efficient method to treat neonatal jaundice while allowing continuous breastfeeding.