title: Glucuronidation of 1-hydroxypyrene by human liver microsomes and human UDP-glucuronosyltransferases UGT1A6, UGT1A7, and UGT1A9: development of a high-sensitivity glucuronidation assay for human tissue.
abstract: Human UDP-glucuronosyltransferases (UGT, EC 2.4.1.17) involved in the biotransformation of pyrene were investigated by a sensitive fluorometric high-performance liquid chromatography (HPLC)method developed for determining activities toward 1-hydroxypyrene. The endpoint metabolite of pyrene, 1-pyrenylglucuronide, is a well-known urinary biomarker for the assessment of human exposure to polycyclic aromatic hydrocarbons. 1-Pyrenylglucuronide was synthesized using rat liver microsomes as biocatalyst. The yield was satisfactory, 22%. 1-Pyrenylglucuronide, identified by (1)H NMR and by electrospray mass spectrometry, was used for method validation and calibration. The HPLC assay was very sensitive with a quantitation limit of 3 pg (8 fmol) for 1-pyrenylglucuronide. The assay was precise, showing a relative standard deviation of 5% or less at 0.1 to 300 microM 1-hydroxypyrene. Only 2 microg of microsomal protein was required for the assay in human liver. The glucuronidation of 1-hydroxypyrene was catalyzed at high rates in microsomes from pooled or three individual liver samples, showing comparable apparent K(m) values. The formation of 1-pyrenylglucuronide was catalyzed by recombinant human UGT1A6, UGT1A7, and UGT1A9, the K(m) values being 45, 12, and 1 microM, respectively. The apparent K(m) values in human liver microsomes, ranging from 6.9 to 8.6 microM, agreed well with these results. The method provides a sensitive tool for measuring extremely low UGT activities and a specific means for assessing interindividual differences in 1-hydroxypyrene-metabolizing UGT activities in human liver and other tissues.
authors.affiliation: Laboratory of Toxicokinetics and Metabolism, Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, FIN-00250 Helsinki, Finland.
authors.forename: L
authors.initials: L
authors.lastname: Luukkanen
authors.forename: J
authors.initials: J
authors.lastname: Mikkola
authors.forename: T
authors.initials: T
authors.lastname: Forsman
authors.forename: P
authors.initials: P
authors.lastname: Taavitsainen
authors.forename: J
authors.initials: J
authors.lastname: Taskinen
authors.forename: E
authors.initials: E
authors.lastname: Elovaara
mesh_terms: D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D020719:Glucuronides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D009153:Mutagens / Q000378:metabolism*; D011721:Pyrenes / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D020719:Glucuronides; D009153:Mutagens; D011721:Pyrenes; D011994:Recombinant Proteins; D014453:Glucuronosyltransferase; C033146:1-hydroxypyrene; D004121:Dimethyl Sulfoxide