Elsevier

Chemico-Biological Interactions

Volume 231, 25 April 2015, Pages 90-97
Chemico-Biological Interactions

Tissue and species differences in the glucuronidation of glabridin with UDP-glucuronosyltransferases

https://doi.org/10.1016/j.cbi.2015.03.001Get rights and content

Highlights

  • HIM displays much higher GA glucuronidation activities than HLM.

  • UGT1A8 plays an important role in glucuronidation of GAin humans.

  • RLM and DLM catalyze GA glucuronidation more effectively than HLM and CyLM.

Abstract

Glabridin (GA) has gained wide application in the cosmetics and food industry. This study was performed to investigate its metabolic inactivation and elimination by glucuronidation by use of liver and intestine microsomes from humans (HLM and HIM) and rats (RLM and RIM), and liver microsomes from cynomolgus monkeys and beagle dogs (CyLM and DLM). Both hydroxyl groups at the C2 and C4 positions of the B ring are conjugated to generate two mono-glucuronides (M1 and M2). HIM, RIM and RLM showed the most robust activity in catalyzing M2 formation with intrinsic clearance values (Clint) above 2000 μL/min/mg, with little measurable M1 formation activity. DLM displayed considerable activity both in M1 and M2 formation, with Clint values of 71 and 214 μL/min/mg, respectively, while HLM and CyLM exhibited low activities in catalyzing M1 and M2 formation, with Clint values all below 20 μL/min/mg. It is revealed that UGT1A1, 1A3, 1A9, 2B7, 2B15 and extrahepatic UGT1A8 and 1A10 are involved in GA glucuronidation. Nearly all UGTs preferred M2 formation except for UGT1A1. Notably, UGT1A8 displayed the highest activity with a Clint value more than 5-fold higher than the other isoforms. Chemical inhibition studies, using selective inhibitors of UGT1A1, 1A9, 2B7 and 1A8, further revealed that UGT1A8 contributed significantly to intestinal GA glucuronidation in humans. In summary, this in vitro study demonstrated large species differences in GA glucuronidation by liver and intestinal microsomes, and that intestinal UGTs are important for the pathway in humans.

Introduction

Glabridin (GA), a polyphenolic isoflavonoid (Fig. 1) extracted from the licorice root, has been used for centuries in Asian and European countries as antidotes, demulcents, and expectorants, as well as flavoring and sweetening agents [21]. GA was reported to display various desired biological activities, including anti-oxidant, anti-inflammatory, anti-tumorigenic, anti-nephritic, anti-bacterial, anti-obesity, skin-whitening, estrogenic-like, and neuro-protective effects [21]. GA and extracts enriched in GA have gained wide application in the cosmetics and food industry [7], [21].

Due to the wide range of applications of GA, several studied have been conducted on its metabolism. The oral availability of the current drug was very low in rats (about 7.5% of the oral dose), which was attributed to P-gp mediated efflux and extensive hepatic glucuronidation [3]. GA can also undergo oxidation by cytochromes P450 enzymes (CYP) with reactive metabolites generated and then inactivate CYP3A4 and CYP2B6 [12]. Since CYP3A4 and CYP2B6 are responsible for metabolism of more than half of all clinically-used drugs, inhibition of these two enzymes by drug–drug interactions may interfere in elimination of a broad spectrum of drugs and induce undesired side effects [12].

Structure–function relationship studies indicate that the two phenol hydroxyl groups in the B ring (Fig. 1) are essential for inactivation of CYP and pharmacological activities [21]. These two hydroxyl groups are potential sites for glucuronidation. Therefore, glucuronidation may exert an impact on the toxic and pharmacological effects of GA. However, detailed information of this metabolic pathway remains poorly understood.

Glucuronidation is mediated by a superfamily called UDP-glucuronosyltransferases (UGTs). UGT expression and even the function of the same homologous isoforms display significant species differences. For example, UGT1A4 and UGT1A9 are not expressed in rats [6], while human and cynomolgus monkey UGT1A6 display different enzymatic properties [8]. Elucidation of the species differences of the glucuronidation will be of great value in selecting an appropriate experimental animal model to conduct the pharmacokinetic, toxicological and pharmacodynamic studies.

UGTs are expressed in various tissues. In humans, since there is abundant expression of many UGTs, liver is usually a major glucuronidation site. However, there are several isoforms that are expressed in extrahepatic tissues. For example, UGT1A8 and UGT1A10 are expressed at the high levels in intestine, but not detected in liver [4], [19]. When the targeted chemical is mainly metabolized by extrahepatic UGTs, the in vivo elimination process may be different from compounds that are mainly metabolized in liver.

The present in vitro study was conducted to investigate GA glucuronidation in microsomes from human liver and intestine, and livers from experimental animals, as well as recombinant UGTs. This study will be helpful for a deeper understanding of human disposition of GA, and provide valuable information in selecting suitable animals for further studies.

Section snippets

Chemical reagents

GA (>98%) and magnolol (>98%) were purchased from the Sichuan Victory company (Chengdu, SC, China). Uridine-5-diphosphoglucuronic acid (trisodium salt) (UDPGA) and alamethicin were purchased from Sigma–Aldrich (St. Louis, MO, USA). Nilotinib (>98%) and fluconazole (99%) was obtained from Alfa Aesar (Shanghai, China). All other reagents were either of HPLC grade or of the highest grade commercially available.

Enzyme sources

Pooled human intestinal microsomes (HIM, n = 10, 7–83 years old, mean 46.7 years old, 20%

Identification of GA Glucuronidation

Incubation of GA with HLM, HIM, RLM, RIM, DLM and CyLM, in the presence of UDPGA, all yielded two metabolite peaks (M1 and M2). In comparison with M2, the formation of M1 was negligible. These two metabolite peaks were absent in the control samples incubated without either microsomes, UDPGA or GA. GA and its glucuronides in assays with HLM and HIM are shown in Fig. 2. Mass spectrometry of M1 and M2 in the negative ionization mode both showed an m/z value of 409 for the deprotonated metabolite,

Discussion

This study investigated the glucuronidation of GA by using recombinant UGTs and microsomes from different tissues of humans and experimental animals. The results indicated that GA glucuronidation could be conjugated by multiple UGTs with remarkable tissue and species variability. GA glucuronidation activity of HIM was demonstrated to be much higher than that of HLM, with the Clint value of more than 100-fold higher (Table 2). Earlier studies revealed that GA glucuronidation occurs at a low rate

Conclusions

The current study indicates that intestinal UGTs, notably UGT1A8 and 1A10, play important roles in GA glucuronidation in humans. Thus, any pharmacological and toxicological of GA effects may depend on activities of these two isoforms. This study also revealed that GA glucuronidation activity varies among species. The rank order of microsomal GA glucuronidation activity is RIM and RLM > HIM > DLM > CyLM and HLM. However, caution should be exercised in attempting to determine the pharmacokinetics and

Conflict of Interest

The authors declare that there are no conflicts of interest.

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Acknowledgments

The authors thank the Startup Project of Doctorial Scientific Research, Anqing Normal University (K05000130011) , Natural Science Foundation of Anhui Educational Commission (AQKJ2014B007) and the 973 Program (2013CB531800) for their support of this work.

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