Identification of cyclosporine A and tacrolimus glucuronidation in human liver and the gastrointestinal tract by a differentially expressed UDP-glucuronosyltransferase: UGT2B7
Introduction
The allogenic transplantation of organs is dependent upon immunosuppression to prevent allograft rejection [1]. The clinical introduction of cyclosporine A (CsA) and tacrolimus (FK506) has led to a significant increase of graft survival and a decrease of morbidity. Both compounds are water insoluble fungal metabolites. CsA is a lipophylic cyclic peptide of 11 residues produced by Tolypocladium inflatum[2], and FK506 is a macrolide lactone derived from Streptomyces tsukubaensis[3]. Although chemically unrelated, both share a remarkably similar mode of action on calcium dependent signal transduction leading to T-cell inhibition [4], [5].
Cyclosporine A and FK506 are usually administered orally and are characterized by an unpredictably variable absorption rate which requires monitoring of drug levels [6]. Extensive metabolism by hepatic cytochrome P450s (CYP) has been demonstrated for both drugs [7], [8], [9]. Extrahepatic CsA metabolism by CYP in human intestine has been demonstrated [10], [11]. The identification of CsA [8] and FK506 [12] glucuronides demonstrates that conjugation also influences the metabolism of these immunosuppressants which has not been analyzed in humans.
UDP-glucuronosyltransferases (UGT) are a superfamily of proteins localized in the endoplasmic reticulum [13]. The UGTs glucuronidate hydrophobic compounds rendering them more water soluble and facilitating subsequent elimination in bile and urine [14]. UGTs are divided into two families, UGT1 and UGT2 [13]. The UGT2 genes are encoded on chromosome 4 [15]. From liver, four UGT2 cDNAs have been cloned and characterized, UGT2B4 [16], UGT2B7 [17], UGT2B10 [18], and UGT2B15 [19], [20]. UGT2B4 is not expressed in small intestine [21], [22]. In contrast, UGT1Agenes are encoded on chromosome 2. A linear array of 12 variable exon-1 cassettes at the 5′ end of the UGT1A locus are followed by 4 common exons (exon 2–5) [23], [24]. Individual UGT1A gene products are generated by a strategy of exon sharing which combines unique exon 1 sequences with the common exons 2–5. This process potentially leads to the formation of nine functional UGT transcripts [23].
UGT1A gene products are expressed in a tissue-specific fashion [25], [26]. The analysis of liver tissue has led to the characterization of UGT1A1 [27], UGT1A3 [28], UGT1A4 [27], UGT1A6 [29], and UGT1A9 RNA [30]. An analysis of the human gastrointestinal tract has identified 3 extrahepatic UGT1A transcripts: UGT1A7, UGT1A8 and UGT1A10 [22], [25], [26], [33], [34]. Tissue-specific regulation of the UGT1A genes represents a biochemical basis for tissue-specific glucuronidation in the human digestive system [25], [35].
In the present study, we examined the glucuronidation of CsA and FK506 using human hepatic, and extrahepatic microsomal protein in addition to 11 recombinant UGT proteins to identify the anatomical site and responsible UGTs of immunosuppressant glucuronidation. Extrahepatic human tissues of the gastrointestinal tract were analyzed to test the hypothesis of mucosal inactivation of CsA and FK506 as a contributor to prehepatic metabolism.
Section snippets
Gastrointestinal tissue samples
Tissue samples were obtained from the Department of Abdominal and Transplant Surgery, Hannover Medical School, Hannover, Germany. Macroscopically and histologically normal liver tissue was obtained from a male patient receiving a liver transplantation for hepatocellular carcinoma. Gastric tissue was from a male patient undergoing gastrectomy for gastric adenocarcinoma. Normal duodenal and jejunal tissue was collected from two male patients receiving a Whipple's procedure for carcinoma of the
Expression and activity of recombinant UGT proteins
Baculovirus expression of UGT cDNAs in Sf9 cells have been optimized to produce abundant catalytically active UGT protein [36], [37]. Previous data and control experiments confirm the presence of the UGT proteins employed in this study by Western blot analysis. In Fig. 1, examples of the activities from expressed UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A9, UGT1A10, as well as liver microsomal protein are shown. The activity of UGT2B4 and UGT2B15 have been previously characterized [20], [22]
Discussion
Recent studies have identified a tissue-specific expression of the human UDP-glucuronosyltransferases (UGT) in gastrointestinal tissues [22], [25], [26], [31], [34]. The epithelia of the gastrointestinal tract represent a metabolically active barrier capable of specific extrahepatic glucuronidation and may serve as protection from cytotoxic and genotoxic compounds [22], [33], [35], [39], [40], [41]. These tissues would therefore be capable of metabolizing orally administered drugs during or
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft Grant STR493/3-1 (to C.P.S.) and USPHS grant GM49135 (to R.H.T). The authors wish to thank Professor J. Klempnauer and Dr R. Raab, Department of Abdominal and Transplant Surgery, for the assistance with tissue sample procurement, and J. Topp, M.S., for technical assistance with the PCR analysis.
References (41)
- et al.
Immunosuppressive therapy
Surg Clin North Am
(1999) - et al.
Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes
Cell
(1991) - et al.
First-pass metabolism of cyclosporin by the gut
Lancet
(1991) - et al.
Isolation of a human YAC contig encompassing a cluster of UGT2 genes and its regional localization to chromosome 4q13
Genomics
(1994) - et al.
Cloning and expression of human liver UDP-glucuronosyltransferase in COS-1 cells, 3,4-catechol estrogens and estriol as primary substrates
J Biol Chem
(1990) - et al.
cDNA cloning and expression of two new members of the human liver UDP- glucuronosyltransferase 2B subfamily
Biochem Biophys Res Commun
(1993) - et al.
UDP-glucuronosyltransferases in human intestinal mucosa
Biochim Biophys Acta
(1998) - et al.
A novel complex locus UGT1 encodes human bilirubin, phenol, and other UDP-glucuronosyltransferase isozymes with identical carboxyl termini
J Biol Chem
(1992) - et al.
Expression of the UDP-glucuronosyltransferase 1A locus in human colon. Identification and characterization of the novel extrahepatic UGT1A8
J Biol Chem
(1998) - et al.
Cloning of two human liver bilirubin UDP-glucuronosyltransferase cDNAs with expression in COS-1 cells
J Biol Chem
(1991)
cDNA cloning and characterization of the human UDP glucuronosyltransferase, UGT1A3
Biochem Biophys Res Commun
Cloning and expression of human UDP-glucuronosyltransferase (UGT) 1A8
Arch Biochem Biophys
Characterization of two UDP glucuronosyltransferases that are predominantly expressed in human colon
Biochem Biophys Res Commun
The human UDP glucuronosyltransferase, UGT1A10, glucuronidates mycophenolic acid
Biochem Biophys Res Commun
UDP-glucuronosyltransferase activity in human liver and colon
Gastroenterology
Autoantibodies against glucuronosyltransferases differ between viral hepatitis and autoimmune hepatitis
Gastroenterology
UDP-glucuronosyltransferases and their role in metabolism and disposition of carcinogens
Adv Pharmacol
Biological effects of cyclosporin A: a new antilymphocytic agent
Agents Actions
FK-506, a novel immunosuppressant isolated from a Streptomyces. I. Fermentation, isolation, and physico-chemical and biological characteristics
J Antibiot (Tokyo)
Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A
Nature
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