Carcinogenesis, Vol 18, 843-849, Copyright © 1997 by Oxford University Press
RS King and MW Duffel
The sulfation of primary N-hydroxy arylamines is a critical intermediate
step in the bioactivation of many carcinogenic arylamines, arylamides 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 arylamines,
N-hydroxyaniline and N-hydroxy-2-aminofluorene, under different oxidative
reaction conditions pertinent to the assay of sulfotransferases.
Furthermore, these compounds, as well as the products of their oxidative
degradation, were examined for their interactions with homogeneous aryl
sulfotransferase (AST) IV. Under reaction conditions where oxidative
degradation of the N-hydroxy arylamines occurred, N-hydroxyaniline and
N-hydroxy-2-aminofluorene produced time-dependent and irreversible
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 showed that the
inhibition of AST IV did require non-enzymatic oxidation of the N-hydroxy
arylamine. Under reaction conditions that prevented the oxidative
degradation of N-hydroxyaniline, this N-hydroxy arylamine was a substrate
for AST IV. Likewise, under similar conditions, 4-chloro-N- hydroxyaniline
was also a substrate for the enzyme. In contrast, no AST IV catalyzed
sulfation of N-hydroxy-2-aminofluorene was detected under conditions that
prevented the oxidation of N-hydroxy-2-aminofluorene. Adequate protection
of these N-hydroxy arylamines from oxidative degradation required the
addition of L-ascorbic acid to reaction mixtures that had also been
degassed and purged with argon. The irreversible inhibition of AST IV
exhibited by these N-hydroxy arylamines, even in reaction mixtures where
attempts were made to limit oxidative degradation by degassing and purging
with argon, emphasized the importance of completely preventing such
degradation when utilizing in vitro assays to assess the potential for an
N-hydroxy arylamine to serve as a substrate for a specific
sulfotransferase.
ARTICLES
Oxidation-dependent inactivation of aryl sulfotransferase IV by primary N-hydroxy arylamines during in vitro assays
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, The University of Iowa, Iowa City 52242, USA.
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