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Research ArticleMetabolism, Transport, and Pharmacogenomics

Identification and Functional Characterization of a Novel UDP-Glucuronosyltransferase 2A1 Splice Variant: Potential Importance in Tobacco-Related Cancer Susceptibility

Ryan T. Bushey and Philip Lazarus
Journal of Pharmacology and Experimental Therapeutics December 2012, 343 (3) 712-724; DOI: https://doi.org/10.1124/jpet.112.198770
Ryan T. Bushey
Departments of Pharmacology (R.T.B., P.L.) and Public Health Sciences (P.L.), Penn State University College of Medicine, Hershey, Pennsylvania
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Philip Lazarus
Departments of Pharmacology (R.T.B., P.L.) and Public Health Sciences (P.L.), Penn State University College of Medicine, Hershey, Pennsylvania
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Abstract

UDP-glucuronosyltransferase (UGT) 2A1 is a respiratory and aerodigestive tract-expressing phase II detoxifying enzyme that metabolizes various xenobiotics including polycyclic aromatic hydrocarbons (PAHs). In the present study, a novel exon 3 deletion splice variant was identified for UGT2A1 (UGT2A1Δexon3). As determined by reverse transcription-polymerase chain reaction (PCR), UGT2A1Δexon3 was shown to be expressed in various tissues including lung, trachea, larynx, tonsil, and colon. The ratio of UGT2A1Δexon3/wild-type UGT2A1 expression was highest in colon (0.79 ± 0.08) and lung (0.42 ± 0.12) as determined by real-time PCR; an antibody specific to UGT2A1 showed splice variant protein (UGT2A1_i2) to wild-type protein (UGT2A1_i1) ratios in the range of 0.5 to 0.9 in these tissues. Using ultra-pressure liquid chromatography, we found that homogenates prepared from UGT2A1_i2-overexpressing human embryonic kidney 293 cells exhibited no glucuronidation activity against PAHs, including benzo[a]pyrene-7,8-dihydrodiol (B[a]P-7,8-diol). An inducible in vitro system was created to determine the effect of UGT2A1_i2 expression on UGT2A1_i1 activity. Increasing UGT2A1_i2 levels resulted in a significant (p < 0.01) decrease in the UGT2A1_i1 Vmax against 1-hydroxy (OH)-pyrene, 3-OH-benzo[a]pyrene, and B[a]P-7,8-diol; no significant changes in KM were observed for any of the three substrates. Coimmunoprecipitation experiments suggested the formation of UGT2A1_i1 and UGT2A1_i2 hetero-oligomers and UGT2A1_i1 homo-oligomers; coexpression of UGT2A1_i1 or UGT2A1_i2 with other UGT1A or UGT2B enzymes caused no change in UGT1A or UGT2B glucuronidation activity. These data suggest that a novel UGT2A1 splice variant regulates UGT2A1-mediated glucuronidation activity via UGT2A1-specific protein-protein interactions, and expression of this variant could play an important role in the detoxification of carcinogens within target tissues for tobacco carcinogenesis.

Footnotes

  • This work was supported by the National Institutes of Health National Institute of Dental and Craniofacial Research [Grant R01-DE13158] (to P.L.); the National Institutes of Health, National Cancer Institute [Grant R01-CA164366] (to P.L.); and the Pennsylvania Department of Health's Health Research Formula Funding Program [Grant 4100038714] (to P.L.).

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

    http://dx.doi.org/10.1124/jpet.112.198770.

  • ↵Embedded Image The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

  • ABBREVIATIONS:

    UGT
    UDP-glucuronosyltransferase
    UDPGA
    UDP-glucuronic acid
    PAH
    polycyclic aromatic hydrocarbon
    PonA
    ponasterone A
    RT
    reverse transcription
    PCR
    polymerase chain reaction
    B[a]P
    benzo[a]pyrene
    B[a]P-7,8-diol
    benzo[a]pyrene-7,8-dihydrodiol
    coIP
    coimmunoprecipitation
    UPLC
    ultra-pressure liquid chromatography
    HEK
    human embryonic kidney
    HRP
    horseradish peroxidase
    HCA
    heterocyclic amine
    N-OH PhIP
    N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine
    TSNA
    tobacco-specific nitrosamine
    NNAL
    4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol
    AU
    arbitrary units.

  • Received July 30, 2012.
  • Accepted September 12, 2012.
  • Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 343 (3)
Journal of Pharmacology and Experimental Therapeutics
Vol. 343, Issue 3
1 Dec 2012
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Research ArticleMetabolism, Transport, and Pharmacogenomics

Characterization of the UGT2A1 Exon 3 Splice Variant

Ryan T. Bushey and Philip Lazarus
Journal of Pharmacology and Experimental Therapeutics December 1, 2012, 343 (3) 712-724; DOI: https://doi.org/10.1124/jpet.112.198770

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Research ArticleMetabolism, Transport, and Pharmacogenomics

Characterization of the UGT2A1 Exon 3 Splice Variant

Ryan T. Bushey and Philip Lazarus
Journal of Pharmacology and Experimental Therapeutics December 1, 2012, 343 (3) 712-724; DOI: https://doi.org/10.1124/jpet.112.198770
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