Expression of human estrogen sulfotransferase in Salmonella typhimurium: differences between hHST and hEST in the enantioselective activation of 1-hydroxyethylpyrene to a mutagen
Introduction
Many carcinogens are detected as mutagens in in vitro test systems (e.g. Ames test), if external activating systems are employed [1]. However, the generated active metabolites can only induce mutations after they have penetrated the cell membrane to reach the target. It is assumed that reactive sulfate conjugates do not readily penetrate the cell membrane, since they are short-lived and ionized. Therefore, it is important to have a test system where the sulfate conjugates are synthesized within the indicator cell 2, 3.
Since xenobiotic-metabolizing sulfotransferases (SULTs) are not endogenously expressed in the standard Salmonella typhimurium strains which are used in the Ames test, SULT-proficient bacterial strains were constructed. In this study two SULT-proficient Salmonella strains, expressing human estrogen sulfotransferase (hEST) and hydroxysteroid sulfotransferase (hHST), respectively, were studied for their ability to activate benzylic alcohols to mutagens.
Section snippets
Synthesis of 1-hydroxymethylpyrene (1-HMP) and 1-hydroxyethylpyrene (1-HEP) enantiomers
1-HMP and 1-HEP (1-(1-pyrenyl)ethanol) were synthesized as described [4]. 1-HEP was separated into its enantiomers by chromatography on Chiralcel (20 μm) using ethanol/hexane (1+9, v/v) as the eluant. The enantiomeric purity of the products was greater than 99.5%. The (−)- and (+)-enantiomers have the S and R configuration at the benzylic position, respectively (Landsiedel et al., manuscript in preparation). An antibody raised against rat estrogen sulfotransferase [5]was generously provided by
Results
Cytosol from TA1538-hEST was probed in a Western blot with a rabbit anti-rEST antibody and a single band at about 33 000 Da was detected (Fig. 1). The mobility of this immunoreactive protein was identical to that observed for a human hepatic enzyme, putatively hEST. We estimate that the expression level in Salmonella is at least 200 times higher than in the liver. In the same blot the cytosol from the SULT-deficient TA1538 strain did not react with the antibody. Cytosol prepared from
Conclusions
1-HMP and both enantiomers of 1-HEP showed mutagenic effects in SULT-proficient S. typhimurium strains, but were not mutagenic in the SULT-deficient TA1538 strain. Thus these compounds were activated by metabolic sulfation and the expression of active SULTs in indicator cells is important for the reliable detection of SULT-dependent mutagens.
The results clearly show the dramatic effect an extension of the side chain of 1-HMP by a methylene group has and how in addition the position of this
Acknowledgements
This work was financially supported by the Deutsche Forschungsgemeinschaft (SFB 302, INK 26) and the Commission of the European Communities (BMH1-CT92-0097 and EV5V-CT94-0410).
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