Vol. 29, Issue 8, 1096-1101, August 2001

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

Leena Luukkanen,¹ Jouni Mikkola, Tarja Forsman, Päivi Taavitsainen, Jyrki Taskinen, and Eivor Elovaara

Laboratory of Toxicokinetics and Metabolism, Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Helsinki, Finland (L.L., J.M., E.E.); Viikki Drug Discovery Technology Center, Department of Pharmacy, University of Helsinki, Finland (L.L., T.F., J.T.); and Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland (P.T.)

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 ¹H 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 µM 1-hydroxypyrene. Only 2 µg 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 µM, respectively. The apparent K_m values in human liver microsomes, ranging from 6.9 to 8.6 µM, 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.