OXIDATION-DEPENDENT INACTIVATION OF ARYL SULFOTRANSFERASE-IV BY PRIMARY N-HYDROXY ARYLAMINES DURING IN-VITRO ASSAYS

The sulfation of primary N-hydroxy arylamines is a critical intermedia te step in the bioactivation of many carcinogenic arylamines, arylamid es and nitroaromatics, However, the study of this reaction in vitro is often complicated by the chemical instability of these molecules, We have examined the stability of two highly purified N-hydroxy arylamine s, N-hydroxyaniline and N-hydroxy-2-aminofluorene, under different oxi dative reaction conditions pertinent to the assay of sulfotransferases . Furthermore, these compounds, as well as the products of their oxida tive degradation, were examined for their interactions with homogeneou s aryl sulfotransferase (AST) IV, Under reaction conditions where oxid ative degradation of the N-hydroxy arylamines occurred, N-hydroxyanili ne and N-hydroxy-2-aminofluorene produced time-dependent and irreversi ble inhibition of AST IV, While this inhibition was not dependent upon the presence of 3'-phosphoadenosine 5'-phosphosulfate in the reaction mixture, analysis of the N-hydroxy arylamines by UV spectroscopy show ed that the inhibition of AST TV did require non-enzymatic oxidation o f the N-hydroxy arylamine, Under reaction conditions that prevented th e oxidative degradation of N-hydroxyaniline, this N-hydroxy arylamine was a substrate for AST IV, Likewise, under similar conditions, 4-chlo ro-N-hydroxyaniline was also a substrate for the enzyme, In contrast, no AST IV catalyzed sulfation of N-hydroxy-2-aminofluorene was detecte d under conditions that prevented the oxidation of N-hydroxy-2-aminofl uorene. Adequate protection of these N-hydroxy arylamines from oxidati ve degradation required the addition of L-ascorbic acid to reaction mi xtures that had also been degassed and purged with argon, The irrevers ible inhibition of AST IV exhibited by these N-hydroxy arylamines, eve n in reaction mixtures where attempts were made to limit oxidative deg radation by degassing and purging with argon, emphasized the importanc e of completely preventing such degradation when utilizing in vitro as says to assess the potential for an N-hydroxy arylamine to serve as a substrate for a specific sulfotransferase.