Isoform‐Selective Probe Substrates for In Vitro Studies of Human UDP‐Glucuronosyltransferases
Introduction
Glucuronidation represents a major metabolic pathway that facilitates efficient detoxification and elimination of potentially harmful xenobiotics (drugs, toxins, carcinogens) and endobiotics (bilirubin, bile acids, hormones, inflammatory mediators) (for a review, see Fisher et al., 2001). This reaction is catalyzed by UDP‐glucuronosyltransferases, a superfamily of enzymes known in humans to contain at least 17 distinct isoforms. UGTs are expressed primarily in the liver, although extrahepatic tissues (particularly kidney and aerodigestive tracts) are also rich sources, reflecting the critical role for these enzymes in protecting against potentially harmful xenobiotics. The majority of UGTs, like other xenobiotic‐metabolizing enzymes, display broad and overlapping substrate specificities. While this provides an effective protective barrier against xenobiotic exposure, it greatly complicates study of the function of individual isoforms within human tissues. Despite this, substantial progress has been made in identifying substrates that are selectively glucuronidated by individual UGT isoforms (see Table I).
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Uses for UGT‐Selective Probes
UGT‐selective probes have a number of important applications in the study of drug glucuronidation. First, they are used to substantiate the identification of UGTs mediating a particular glucuronidation reaction in human tissues via activity correlation analysis. For example, we have verified the role of UGT2B15 in S‐lorazepam glucuronidation by human liver by showing significant correlation between this activity and S‐oxazepam glucuronidation activities (a UGT2B15 probe) measured in the same
Evaluation of UGT Probe Selectivity
UGT substrates that are either being used as UGT‐selective probes or show potential for this purpose based on experimental data from the literature (Bernard 2004, Court 2002, Court 2003, Dehal 2001, Gagne 2002, Innocenti 2001, Kasai 2005, Krishnaswamy 2003, Krishnaswamy 2004, Nakajima 2002, Nishiyama 2002, Patten 2001, Turgeon 2001, Watanabe 2003, Zhang 2003) or unpublished work from this laboratory are listed in Table I. Three criteria that we have found useful in evaluating the potential
Incubation Protocol
Details regarding incubation conditions specific to each substrate are given in Table II. The following description assumes a 100‐μl final incubation volume, although it can be scaled to other volumes. Substrates, glucuronide standards, and internal standards are conveniently prepared by dissolving in methanol (some additional water may be needed to dissolve glucuronides) and stored at −20°. Substrate is added to each incubation tube (0.5‐ or 1.5‐ml polypropylene microcentrifuge) and dried down
Concluding Remarks
Finally, it should be pointed out that while the particular probes described here have proven of substantial utility in studies in our laboratory (and elsewhere), based on the evidence provided in Table I not all of these probes are considered ideal in terms of isoform selectivity. Moreover, isoform‐selective substrates have not yet been identified for the important extrahepatic UGT isoforms, such as UGTs 1A7, 8, and 10. Given the growing number of drugs and other compounds that are being
Acknowledgments
This work was supported in part by Grant GM‐61834 from the National Institutes of Health (Bethesda, MD) and by Pfizer Global Research and Development (Ann Arbor, MI).
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