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

Human UDP-glucuronosyltransferases (UGT, EC 2.4.1.17) involved in the biotr ansformation of pyrene were investigated by a sensitive fluorometric high-p erformance liquid chromatography (HPLC) method developed for determining ac tivities toward 1-hydroxypyrene. The endpoint metabolite of pyrene, 1-pyren ylglucuronide, is a well-known urinary biomarker for the assessment of huma n exposure to polycyclic aromatic hydrocarbons, 1-Pyrenylglucuronide was sy nthesized using rat liver microsomes as biocatalyst. The yield was satisfac tory, 22%. 1-Pyrenylglucuronide, identified by H-1 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 devi ation of 5% or less at 0.1 to 300 muM 1-hydroxypyrene. Only 2 mug of micros omal protein was required for the assay in human liver. The glucuronidation of 1-hydroxypyrene was catalyzed at high rates in microsomes from pooled o r three individual liver samples, showing comparable apparent K-m values. T he formation of 1-pyrenylglucuronide was catalyzed by recombinant human UGT 1AG, UGT1A7, and UGT1A9, the K-m values being 45, 12, and 1 muM, respective ly. The apparent K-m values in human liver microsomes, ranging from 6.9 to 8.6 muM, agreed well with these results. The method provides a sensitive to ol for measuring extremely low UGT activities and a specific means for asse ssing interindividual differences in 1-hydroxypyrene-metabolizing UGT activ ities in human liver and other tissues.