In addition to serving as a
biomarker of oxidative/nitrative stress, elevated levels of
nitrotyrosine have been shown to cause DNA damage or trigger apoptosis. Whether the body is equipped with mechanisms for protecting against the potentially harmful
nitrotyrosine remains unknown. The present study was designed to investigate the possibility that sulfation serves as a pathway for the metabolism/regulation of
nitrotyrosine. Using metabolic labelling,
nitrotyrosine O-[35S]
sulfate was found to be produced and released into the medium of HepG2 human
hepatoma cells labelled with [35S]
sulfate in the presence of
nitrotyrosine. To identify the
enzyme(s) responsible for
nitrotyrosine sulfation, a systematic study of all eleven known human cytosolic SULTs (
sulfotransferases) was performed. Of the 11
enzymes tested, only
SULT1A3 displayed sulfating activity toward
nitrotyrosine. The pH-dependence and kinetic constants of
SULT1A3 with
nitrotyrosine or
dopamine as substrate were determined. To examine whether the sulfation of
nitrotyrosine occurs in the context of cellular physiology, HepG2 cells labelled with [35S]
sulfate were treated with SIN-1 (morpholinosydnonimine), a
peroxynitrite generator. Increments of
nitrotyrosine O-[35S]
sulfate were detected in the medium of HepG2 cells treated with higher concentrations of SIN-1. To gain insight into the physiological relevance of
nitrotyrosine sulfation, a time-course study was performed using [3H]
tyrosine-labelled HepG2 cells treated with SIN-1. The findings confirm that the bulk of free [3H]
nitrotyrosine inside the cells was present in the unconjugated form. The proportion of sulfated [3H]
nitrotyrosine increased dramatically in the medium over time, implying that sulfation may play a significant role in the metabolism of free
nitrotyrosine.