Sulphonation of N-hydroxy-2-acetylaminofluorene by human dehydroepiandrosterone sulphotransferase.
1. The aim was to determine which human recombinant sulphotransferase (ST) isoform(s) were responsible for the sulphonation and, thus, potential further bioactivation of the classical hepatic procarcinogen N-hydroxy-2-acetylaminofluorene (N-OH-2AAF). 2. N-OH-2AAF was incubated together with the cosubstrate 3'-phosphoadenosine-5'-phosphosulphate (PAPS) and either human liver cytosol or recombinant P-form phenolsulphotransferase (P-PST), M-form PST, dehydroepiandrosterone-ST (DHEA-ST) or oestrogen ST (EST). Formation of 3'-phosphoadenosine-5'-phosphate (PAP) from PAPS, measured by HPLC, was used as the assay for determination of sulphoconjugation rates. 3. The liver cytosol produced a 100% increase in PAP formation in the presence of 200 microM N-OH-2AAF as compared with baseline levels (p < 0.01), corresponding to a rate of 19 pmol/min/mg protein. Recombinant P-PST, however, was without effect. This is in contrast to previous suggestions using crude enzyme preparations. Like P-PST, recombinant M-PST and EST did not sulphonate N-OH-2AAF. On the other hand, recombinant DHEA-ST produced a 161% increase in PAP formation in the presence of 200 microM N-OH-2AAF as compared with baseline values (p < 0.001). 4. Kinetic studies of N-OH-2AAF sulphonation by DHEA-ST and human liver cytosol gave similar apparent Kms. Interestingly, the Vmax for N-OH-2AAF sulphonation by DHEA-ST was very similar to that of DHEA, the natural substrate for DHEA-ST. 5. This is the first paper to demonstrate the involvement of the human DHEA-ST in the sulphonation of an N-hydroxylated aromatic amide carcinogen.
Lewis
A J
AJ
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston 29425, USA.
Otake
Y
Y
Walle
U K
UK
Walle
T
T
D000244:Adenosine Diphosphate / Q000378:metabolism; D002273:Carcinogens / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D003687:Dehydroepiandrosterone / Q000737:chemistry*; D006801:Humans; D006881:Hydroxyacetylaminofluorene / Q000378:metabolism*; D007527:Isoenzymes; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry*; D015238:Sulfotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000494:pharmacology; D013463:Sulfuric Acid Esters / Q000378:metabolism
D002273:Carcinogens; D007527:Isoenzymes; D011994:Recombinant Proteins; D013463:Sulfuric Acid Esters; D003687:Dehydroepiandrosterone; D006881:Hydroxyacetylaminofluorene; D000244:Adenosine Diphosphate; C010840:adenosine 3'-phosphate-5'-phosphate; D015238:Sulfotransferases; C019998:dehydroepiandrosterone sulfotransferase