Identification of a New Reactive
Metabolite of Pyrrolizidine
Alkaloid Retrorsine: (3<i>H</i>‑Pyrrolizin-7-yl)methanol
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Abstract
Pyrrolizidine alkaloids (PAs) such
as retrorsine are common food
contaminants that are known to be bioactivated by cytochrome P450
enzymes to putative hepatotoxic, genotoxic, and carcinogenic metabolites
known as dehydropyrrolizidine alkaloids (DHPs). We compared how both
electrochemical (EC) and human liver microsomal (HLM) oxidation of
retrorsine could produce short-lived intermediate metabolites; we
also characterized a toxicologically important metabolite, (3<i>H</i>-pyrrolizin-7-yl)methanol. The EC cell was coupled online
or offline to a liquid chromatograph/mass spectrometer (LC/MS), whereas
the HLM oxidation was performed in 100 mM potassium phosphate (pH
7.4) in the presence of NADPH at 37 °C. The EC cell oxidation
of retrorsine produced 12 metabolites, including dehydroretrorsine
(<i>m</i>/<i>z</i> 350, [M + H<sup>+</sup>]),
which was degraded to a new reactive metabolite at <i>m</i>/<i>z</i> 136 ([M + H<sup>+</sup>]). The molecular structure
of this small metabolite was determined using high-resolution mass
spectrometry and NMR spectroscopy followed by chemical synthesis.
In addition, we also identified another minor but reactive metabolite
at <i>m</i>/<i>z</i> 136, an isomer of (3<i>H</i>-pyrrolizin-7-yl)methanol. Both (3<i>H</i>-pyrrolizin-7-yl)methanol
and its minor isomer were also observed after HLM oxidation of retrorsine
and other hepatotoxic PAs such as lasiocarpine and senkirkin. In the
presence of reduced glutathione (GSH), each isomer formed identical
GSH conjugates at <i>m</i>/<i>z</i> 441 and <i>m</i>/<i>z</i> 730 in the negative ESI-MS. Because
(3<i>H</i>-pyrrolizine-7-yl)methanol) and its minor isomer
subsequently reacted with GSH, it is concluded that (3<i>H</i>-pyrrolizin-7-yl)methanol may be a common toxic metabolite arising
from PAs