Characterization of Hepatic and Intestinal Glucuronidation of Magnolol: Application of the Relative Activity Factor Approach to Decipher the Contributions of Multiple UDP-Glucuronosyltransferase Isoforms

Zhu, Liangliang; Ge, Guang-Bo; Zhang, Hongbo; Liu, Hui‐Xin; He, Guiyuan; Liang, Sisi; Zhang, Yanyan; Fang, Zhong‐Ze; Dong, Peipei; Finel, Moshe; Yang, Ling

doi:10.1124/dmd.111.042192

Cited by 59 publications

(60 citation statements)

References 32 publications

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“…Apart from these reasons, it is notable that the activities of recombinant UGTs do not directly and quantitatively mirror the actual UGT activities in human tissues. In this regard, the relative activity factor approach (Crespi and Miller, 1999), which uses the ratio of activity of tissue microsomes and recombinant enzymes, would be useful to estimate the contributions of individual UGTs to a given metabolic pathway in tissue microsomes, as recent studies reported (Kato et al, 2012;Zhu et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

Preparation of a Specific Monoclonal Antibody against Human UDP-Glucuronosyltransferase (UGT) 1A9 and Evaluation of UGT1A9 Protein Levels in Human Tissues

Oda¹,

Nakajima²,

Hatakeyama³

et al. 2012

Drug Metab Dispos

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ABSTRACT:Glucuronidation is a major detoxification pathway of drugs and xenobiotics that are catalyzed by the UDP-glucuronosyltransferase (UGT) superfamily. Determination of the protein levels of the individual UGT isoforms in human tissues is required for the successful extrapolation of in vitro metabolic data to in vivo clearance. Most previous studies evaluating UGT isoform expression were limited to the mRNA level because of the high degree of amino acid sequence homology between UGT isoforms that has hampered the availability of isoform-specific antibodies. In this study, we generated a peptide-specific monoclonal antibody against human UGT1A9. We demonstrated that this antibody does not cross-react with the other UGT1A isoforms including UGT1A7, UGT1A8, and UGT1A10 and shows a high degree of amino acid sequence similarity with UGT1A9. Using this antibody, we found that UGT1A9 protein is expressed in the kidney and the liver but not in the jejunum or the ileum, consistent with previous reports of mRNA expression. In a panel of 20 individual human livers, the UGT1A9 protein levels exhibited 9-fold variability. It is noteworthy that the relative UGT1A9 protein levels were not correlated with the UGT1A9 mRNA level (r ‫؍‬ ؊0.13), like other UGT isoforms reported previously, suggesting the importance of evaluating UGT isoform expression at protein levels. In conclusion, we generated a specific monoclonal antibody against UGT1A9 and evaluated the distribution and relative expression levels of the UGT1A9 protein in human tissues. This antibody may serve as a useful tool for further studies of UGT1A9 to evaluate its physiological, pharmacological, and toxicological roles in human tissues.

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Section: Discussionmentioning

confidence: 99%

Preparation of a Specific Monoclonal Antibody against Human UDP-Glucuronosyltransferase (UGT) 1A9 and Evaluation of UGT1A9 Protein Levels in Human Tissues

Oda¹,

Nakajima²,

Hatakeyama³

et al. 2012

Drug Metab Dispos

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“…20 A Shimpack XR-ODS (50.0 mm × 2.0 mm i.d., 2.2 μm, Shimadzu) analytical column with an ODS guard column (5 mm × 2.0 mm i.d., 2.2 μm, Shimadzu) was used to separate DES and its glucuronide. The mobile phase consisted of CH 3 CN (A) and Millipore water containing 0.2% formic acid (B).…”

Section: ■ Experimental Proceduresmentioning

confidence: 99%

Characterization of UDP-Glucuronosyltransferases Involved in Glucuronidation of Diethylstilbestrol in Human Liver and Intestine

Zhu

Liu

et al. 2012

Chem. Res. Toxicol.

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Diethylstilbestrol (DES), a synthetic estrogen, is famous for its carcinogenic effects. Human exposure to this compound can occur frequently through dietary ingestion and medical treatment. Glucuronidation has been demonstrated to be a predominant metabolic pathway for DES in human. Therefore, glucuronidation metabolism may have a significant impact on its toxicities, and it is essential to clarify this metabolic pathway. Accordingly, this in vitro study is designed to characterize the UGTs involved in DES glucuronidation and, furthermore, to identify the roles of individual isoforms in the reaction in liver and intestine. Human liver microsomes (HLM) displayed much higher potential for DES glucuronidation than human intestinal microsomes (HIM). The intrinsic clearances in HLM and HIM were demonstrated to be 459 and 14 μL/min/mg protein, respectively. Assays with recombinant UGTs demonstrated that UGT1A1, -1A3, -1A8, and -2B7 could catalyze DES glucuronidation, among which UGT2B7 showed the highest affinity. Chemical inhibitors of UGT2B7 and UGT1A1/1A3 both displayed similar inhibition against the reaction in UGT2B7 and HLM. In addition, DES glucuronidation in individual HLM exhibited a large individual variability and strongly correlated to UGT2B7 activity. All evidence indicates that UGT2B7 may act as a major enzyme responsible for DES glucuronidation in human liver. For HIM, both UGT2B7 inhibitor and UGT1A1/1A3/1A8 inhibitor exerted moderate inhibition. It is suggested that although UGT2B7 contributes to DES glucuronidation in intestine, other UGTs may contribute equally. In summary, this study characterizes human UGTs involved in DES glucuronidation in human liver and intestine, which may be helpful for further study about DES-related toxicities. Figure 1), a synthetic nonsteroid estrogen, is famous for its carcinogenicity. Human exposure to this compound can occur directly or indirectly through dietary ingestion and medical treatment. 1−4 DES was once prescribed widely to millions of pregnant women in false hopes of preventing miscarriage and other pregnancy complications in the United States. 1 It is now still accepted as an efficient therapy for the treatment of prostate and breast cancers. 2,3 Aside from its wide clinical applications, DES has found widespread application as a growth promoter in feeding cattle, sheep, and poultry. 4 It was estimated that in 1971 alone as much as 27600 kg of DES was used in livestock feeding in the United States. 5 After widespread and extensive exposures to DES, numerous serious health problems have been encountered. DES was established to cause the rare clear cell adenocarcinoma (CCAC) of the vagina and cervix in "DES daughters" (who are exposed to DES before birth). 6 In addition to CCAC, these daughters were found to suffer from malformations of their genital tracts, which can result in severe pregnancy and fertility problems. 7−9 Just like DES daughters, DES sons were also found to be prone to DES damage in the genitalia. 10 In company with daughters and so...

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“…Two highly selective UGT1A9 inhibitors (niflumic acid and magnolol) were used because of their potent inhibitory effects on UGT1A9-mediated glucuronidation in both recombinant enzymes and in human tissue preparations, as well as their minor effects on the activities of other UGTs (Miners et al, 2011;Zhu et al, 2012). The inhibitory effects of niflumic acid (10 mM) and magnolol (1 mM) on the glucuronidation of esculetin and its derivatives in pooled HLM, UGT1A6, and UGT1A9 were evaluated.…”

Section: Resultsmentioning

confidence: 99%

“…Two selective UGT1A9 inhibitors (niflumic acid or magnolol) were used to explore the contribution of UGT1A9 in the glucuronidation of each 6,7-DHC (Miners et al, 2011;Zhu et al, 2012). Each substrate (esculetin, 4-ME, 4-PE, or 4-HME) was incubated with pooled HLM, UGT1A6, or UGT1A9 in the absence or presence of niflumic acid (10 mM) or magnolol (1 mM).…”

Section: Methodsmentioning

confidence: 99%

Structural Modifications at the C-4 Position Strongly Affect the Glucuronidation of 6,7-Dihydroxycoumarins

Xia

Wang

et al. 2015

Drug Metab Dispos

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Esculetin (6,7-dihydroxycoumarin) and its C-4 derivatives have multiple pharmacologic activities, but the poor metabolic stability of these catechols has severely restricted their application in the clinic. Glucuronidation plays important roles in catechols elimination, although thus far the effects of structural modifications on the metabolic selectivity and the catalytic efficacy of the human UDPglucuronosyltransferase (UGT) enzymes remain unclear. This study was aimed at exploring the structure-glucuronidation relationship of esculetin and its C-4 derivatives, including 4-methyl esculetin, 4-phenyl esculetin, and 4-hydroxymethyl esculetin as well as 4-acetic acid esculetin. It was achieved by identifying the main human UGTs responsible for the different reactions and by careful characterization of the reactions kinetics. These catechols, with the exception of 4-acetic acid esculetin, are selectively metabolized to the corresponding 7-O-glucuronides. UGT1A6 and UGT1A9 are the two major UGTs involved in the 7-O-glucuronidation of 4-methyl esculetin and esculetin. UGT1A6 was the major contributor for 7-O-glucuronidation of 4-hydroxymethyl esculetin, and UGT1A9 played a significant role in the 7-O-glucuronidation of 4-phenyl esculetin. The results of the kinetic analyses revealed that the K m values of the compounds, in both UGT1A9 and human liver microsomes, decreased with increasing hydrophobicity of the C-4 substitutions. The outcome of this was that C-4 hydrophobic and hydrophilic groups on 6,7-dihydroxycoumarin exhibited contrasting effects on UGT affinity. All of these findings provide helpful guidance for further structural modification of 6,7-dihydroxycoumarins with improved metabolic stability.

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Characterization of Hepatic and Intestinal Glucuronidation of Magnolol: Application of the Relative Activity Factor Approach to Decipher the Contributions of Multiple UDP-Glucuronosyltransferase Isoforms

Cited by 59 publications

References 32 publications

Preparation of a Specific Monoclonal Antibody against Human UDP-Glucuronosyltransferase (UGT) 1A9 and Evaluation of UGT1A9 Protein Levels in Human Tissues

Preparation of a Specific Monoclonal Antibody against Human UDP-Glucuronosyltransferase (UGT) 1A9 and Evaluation of UGT1A9 Protein Levels in Human Tissues

Characterization of UDP-Glucuronosyltransferases Involved in Glucuronidation of Diethylstilbestrol in Human Liver and Intestine

Structural Modifications at the C-4 Position Strongly Affect the Glucuronidation of 6,7-Dihydroxycoumarins

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