Abstract
Gunn rats glucuronidate acetaminophen (APAP) at reduced rates and show increased susceptibility to APAP-induced hepatotoxicity. This defect is presumed to involve UDP-glucuronosyltransferase (UGT) 1A6, which is nonfunctional in Gunn rats, but it is currently unclear whether other 1A family members are also involved. In humans, two 1A isoforms are known to be active (1A6 and 1A9) but 1A6 form has a 25-fold lower apparent Km (2 mM). Rat liver microsomal APAP UGT activity is induced by in vivo treatment with β-naphthoflavone or oltipraz, an effect correlating with induction of 1A6 and 1A7. To address a possible role of 1A7 in APAP glucuronidation relative to other 1A forms, cDNAs encoding UGTs 1A1, 1A5, 1A6, 1A7, and 1A8 were expressed in human embryonic kidney cells and the contents of expressed enzyme in prepared membrane fractions determined by quantitative immunoblotting. At 2.5 mM APAP, 1A7 showed the highest specific activity (2.8 nmol/min/nmol 1A7 protein), followed by 1A6 (1.1 nmol/min/nmol), and 1A8 (0.27 nmol/min/nmol). 1A1 and 1A5 were essentially inactive. Kinetic comparisons indicated 1A7 had a similar apparent Km as 1A6 (4.7 versus 3.9 mM, respectively) but a 2.4-fold higher catalytic activity. These data suggest that in rats, 1A7 plays a major role in APAP glucuronidation and contributes to protection against APAP-induced hepatotoxicity. The involvement of other UGTs besides 1A6 is further underscored by the presence of significant residual APAP-glucuronidating activity by Gunn rat hepatocytes, indicating the activity of an unknown UGT2 family member.
Footnotes
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This work was supported by the National Institute of Environmental Health Science Grant ES07762-06 (to J.K.R.). D.J.A. was supported, in part, by an Institutional Training Grant in Toxicological Sciences (ES078087) and a gift from the American Liver Foundation in memory of Louis John Bastone.
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↵2 Bock et al. (1993) referred to the 3-methylcholanthrene-inducible phenol UGT as the UGT1A1 form. Under the current nomenclature guidelines (Mackenzie et al., 1997), this form is now designated UGT1A6.
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↵3 The nucleotide sequences for the cDNA clones were submitted to the GenBank database and assigned accession numbersAF461734–38.
- Abbreviations used are::
- APAP
- acetaminophen
- UGT
- uridine 5′-diphosphoglucuronosyl transferase
- BNF
- β-naphthoflavone
- HEK
- human embryonic kidney
- HPLC
- high-performance liquid chromatography
- Received July 31, 2001.
- Accepted December 5, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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