Elucidation of xenoestrogen metabolism by non-targeted, stable isotope-assisted mass spectrometry in breast cancer cells

Environmental exposure to xenoestrogens, i.e., chemicals that imitate the hormone 17 beta-estradiol, has the potential to influence hormone homeostasis and action. Detailed knowledge of xenobiotic biotransformation processes in cell models is key when transferring knowledge learned from in vitro models to in vivo relevance. This study elucidated the metabolism of two naturally-occurring phyto- and mycoestrogens; namely genistein and zearalenone, in an estrogen receptor positive breast cancer cell line (MCF-7) with the aid of stable isotopeassisted metabolomics and the bioinformatic tool MetExtract II. Metabolism was studied in a time course experiment after 2 h, 6 h and 24 h incubation. Twelve and six biotransformation products of zearalenone and genistein were detected, respectively, clearly demonstrating the abundant xenobiotic biotransformation capability of the cells. Zearalenone underwent extensive phase-I metabolism resulting in alpha-zearalenol (alpha-ZEL), a molecule known to possess a significantly higher estrogenicity, and several phase-II metabolites (sulfo- and glycoconjugates) of the native compound and the major phase I metabolite alpha-ZEL. Moreover, potential adducts of zearalenone with a vitamin and several hydroxylated metabolites were annotated. Genistein metabolism resulted in sulfation, combined sulfation and hydroxylation, acetylation, glucuronidation and unexpectedly adduct formation with pentose- and hexose sugars. Kinetics of metabolite formation and subsequent excretion into the extracellular medium revealed a time-dependent increase in most biotransformation products. The untargeted elucidation of biotransformation products formed during cell culture experiments enables an improved and more meaningful interpretation of toxicological assays and has the potential to identify unexpected or unknown metabolites.

Automated summary

This study utilized stable isotope-assisted metabolomics to investigate the metabolism of two naturally-occurring phyto- and mycoestrogens in a cell model, revealing the abundant xenobiotic biotransformation capability of the cells and the time-dependent increase in most biotransformation products. The untargeted elucidation of biotransformation products formed during cell culture experiments enables a more meaningful interpretation of toxicological assays and has the potential to identify unexpected or unknown metabolites.