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 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 apparentKm values. The formation of 1-pyrenylglucuronide was catalyzed by recombinant human UGT1A6, UGT1A7, and UGT1A9, the Km values being 45, 12, and 1 μM, respectively. The apparent Km 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.
Footnotes
-
↵1 Present address: Viikki Drug Discovery Technology Center, Department of Pharmacy, P.O. Box 56 (Viikinkaari 5E), FIN-00014 University of Helsinki, Finland.
-
This work was supported by the Commission of the European Communities, Biomed 2 program (BMH4-CT97-2621).
- Abbreviations used are::
- 1-HP
- 1-hydroxypyrene
- PAH
- polycyclic aromatic hydrocarbon
- UGT
- UDP-glucuronosyltransferase
- UDPGA
- UDP-glucuronic acid
- HPGA
- 1-pyrenylglucuronide
- DMSO
- dimethyl sulfoxide
- BSA
- bovine serum albumin
- HPLC
- high-performance liquid chromatography
- SFV
- Semliki Forest virus
- RSD
- relative standard deviation
- Received January 9, 2001.
- Accepted April 26, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|