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.
